Anti-cd38 antibodies and fusions to attenuated interferon alpha-2b

ABSTRACT

Antibodies that specifically bind to CD38, as well as constructs comprising such antibodies fused to attenuated interferon alpha-2B proteins are provided. Anti-CD38-attenuated interferon alpha-2b fusion constructs may be used to inhibit proliferation in cancerous cells that express both CD38 and the receptor for IFN-alpha2b, as well as to induce apoptosis in such cells. Inhibition of proliferation and induction of apoptosis in cancerous cells may serve as the basis for the treatment of the underlying cancer.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.14/922,282, filed Oct. 26, 2015, which is a continuation ofInternational Application No. PCT/US2013/038659, filed on Apr. 29, 2013,the disclosure of each of which are hereby incorporated by referencetheir entirety.

REFERENCE TO A SEQUENCE LISTING

This application includes a Sequence Listing submitted electronically asa text file named Anti-CD38_Antibodies_ST25, created on Apr. 29, 2013with a size of 462,000 bytes. The Sequence Listing is incorporated byreference herein.

FIELD OF THE DISCLOSURE

This disclosure relates generally to the field of antibody engineering.More specifically, this disclosure relates to antibodies that bindspecifically to CD38, as well as constructs comprising such antibodiesand attenuated interferon-alpha ligands, and methods of treatment usingthese constructs. In these constructs, the antibodies direct the ligandsto cells that express both CD38 and receptors for the ligands, and theattenuated interferon-alpha reduces interferon signaling in cells thatdo not express CD38.

BACKGROUND OF THE DISCLOSURE

Various publications, including patents, published applications,technical articles, scholarly articles, and gene or protein accessionnumbers are cited throughout the specification. Each of these materialsis incorporated by reference herein, in its entirety and for allpurposes.

CD38 is a 46 kDa type II transmembrane glycoprotein. It has a shortN-terminal cytoplasmic tail of 20 amino acids, a single transmembranehelix and a long extracellular domain of 256 amino acids. It isexpressed on the surface of many immune cells including CD4 and CD8positive T cells, B cells, NK cells, monocytes, plasma cells and on asignificant proportion of normal bone marrow precursor cells. In someinstances, the expression of CD38 in lymphocytes may be dependent on thedifferentiation and activation state of the cell, for example, resting Tand B cells may be negative while immature and activated lymphocytes maybe predominantly positive for CD38 expression. CD38 mRNA expression hasbeen detected in non-hemopoeitic organs such as the pancreas, brain,spleen and liver (Koguma, T. (1994) Biochim. Biophys. Acta 1223:160).

CD38 is a multifunctional ectoenzyme that is involved in transmembranesignaling and cell adhesion. It is also known as cyclic ADP ribosehydrolase because it can transform NAD⁺ and NADP⁺ into cADPR, ADPR andNAADP, depending on extracellular pH. These products induceCa²⁺-mobilization inside the cell, which can lead to tyrosinephosphorylation and activation of the cell. CD38 is also a receptor thatcan interact with a ligand, CD31. Activation of receptor via CD31 leadsto intracellular events including Ca²⁺ mobilization, cell activation,proliferation, differentiation and migration.

CD38 is expressed at high levels on multiple myeloma cells, in mostcases of T- and B-lineage acute lymphoblastic leukemias, some acutemyelocyticleukemias, follicular center cell lymphomas and Tlymphoblastic lymphomas. CD38 is also expressed on B-lineage chroniclymphoblastic leukemia (B-CLL) cells. In some cases, B-CLL patientspresenting with a CD38+ clone are characterized by an unfavorableclinical course with a more advanced stage of disease, poorresponsiveness to chemotherapy and shorter survival time. The use ofantibodies to CD38 has been proposed for the treatment ofCD38-expressing cancers and hematological malignancies. It may thereforebe advantageous to provide alternative antibodies to CD38 which havedesirable manufacturing, stability and immunogenic properties.

Numerous peptide and polypeptide ligands have been described to functionby interacting with a receptor on a cell surface, and therebystimulating, inhibiting, or otherwise modulating a biological response,usually involving signal transduction pathways inside the cell thatbears the said receptor. Examples of such ligands include peptide andpolypeptide hormones, cytokines, chemokines, growth factors, andapoptosis-inducing factors.

Due to the biological activities of such ligands, many have potentialuses as therapeutics. Several peptide or polypeptide ligands have beenapproved by regulatory agencies as therapeutic products including, forexample, human growth hormone, insulin, interferon (IFN)-alpha2b,IFN-alpha2a, IFNβ, erythropoietin, G-CSF and GM-CSF.

While these and other ligands have demonstrated potential in therapeuticapplications, they may also exhibit toxicity when administered to humanpatients. One reason for toxicity is that most of these ligands triggerreceptors on a variety of cells, including cells other than those thatmediate the desired therapeutic effect. A consequence of such “offtarget” activity of ligands is that many ligands are currently notsuitable for use as therapeutic agents because the ligands cannot beadministered at sufficiently high dosages to produce maximal or optimaltherapeutic effects on the target cells which mediate the therapeuticeffect.

For example it has been known since the mid-1980's that interferons, inparticular IFN-alpha, are able to increase apoptosis and decreaseproliferation of certain cancer cells. IFN-alpha has been approved bythe FDA for the treatment of several cancers including melanoma, renalcell carcinoma, B cell lymphoma, multiple myeloma, chronic myelogenousleukemia (CML) and hairy cell leukemia. A direct effect of IFN-alpha onthe tumor cells is mediated by the IFN-alpha binding directly to thetype I IFN receptor on those cells and stimulating apoptosis, terminaldifferentiation or reduced proliferation. A further indirect effect ofIFN-alpha on non-cancer cells is to stimulate the immune system, whichmay produce an additional anti-cancer effect by causing the immunesystem to reject the tumor.

These biological activities are mediated by type I interferon receptorson the surface of the cancer cells which, when stimulated, initiatevarious signal transduction pathways leading to reduced proliferationand/or the induction of terminal differentiation or apoptosis. The typeI interferon receptor is, however, also present on most non-cancerouscells. Activation of this receptor on non-cancerous cells by IFN-alphacauses the expression of numerous pro-inflammatory cytokines andchemokines, leading to toxicity and untoward effects. Such toxicity maycause severe flu-like symptoms, which prevents the dosing of IFN-alphato a subject at levels that exert the maximum anti-proliferative andpro-apoptotic activity on the cancer cells.

When IFN-alpha2b is used to treat multiple myeloma, its utility resides,at least in part, in its binding to type I interferon receptors on themyeloma cells, which in turn triggers apoptosis and/or reducedproliferation and hence limits disease progression. Unfortunately,however, this IFN also binds healthy cells within the body, triggering avariety of other cellular responses, some of which are harmful.

A publication by Ozzello (Breast Cancer Research and Treatment25:265-76, 1993) describes chemically conjugating human IFN-alpha to atumor-targeting antibody, thereby localizing the direct inhibitoryactivity of IFN-alpha to the tumor as a way of reducing tumor growthrates, and demonstrated that such conjugates have anti-tumor activity ina xenograft model of a human cancer. The mechanism of the observedanti-cancer activity was attributed to a direct effect of IFN-alpha onthe cancer cells, since the human IFN-alpha used in the experiments didnot interact appreciably with the murine type I IFN receptor, whichcould have led to an indirect anti-cancer effect. Because of this lackof binding of the human IFN-alpha to the murine cells, the toxicity ofthe antibody-IFN-alpha conjugate relative to free INF-alpha was notassessed.

Antibodies and IFN-alpha may also be connected together in the form of afusion protein. For example, WO 01/97844 describes a direct fusion ofhuman IFN-alpha to the C-terminus of the heavy chain of an IgG specificfor the tumor antigen CD20.

In general, IFN may be targeted to cancer cells. While this approach mayresult in an increase in activity of the IFN against cancer cells, itdoes not completely address the issue of undesired activity of the IFNon healthy cells. Fusing IFN-alpha to the C-terminus of the heavy chainof an IgG may prolong the half-life of the IFN alpha leading toundesirable adverse events. Accordingly, there exists a need to decreaseoff-target activity of ligand-based drugs, while retaining the“on-target” therapeutic effect of such ligands.

SUMMARY OF THE DISCLOSURE

The disclosure features new anti-CD38 antibodies and constructscomprising an anti-CD38 antibody and attenuated IFN-alpha. Theantibodies, which comprise one or a plurality of mutations in theirheavy and/or light chain variable regions retain the ability tospecifically bind to CD38, including CD38 expressed on the surface ofcells. The antibodies may be fused, for example, to an attenuated formof interferon alpha to form an anti-CD38 antibody-attenuated interferonfusion construct.

In some aspects, an isolated antibody that binds specifically to CD38comprises a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 559 and a light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 664. In some aspects, an isolatedantibody that binds specifically to CD38 comprising a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 665 anda light chain variable region comprising the amino acid sequence of SEQID NO: 666. In some aspects, an isolated antibody that bindsspecifically to CD38 comprises a heavy chain variable region comprisingthe amino acid sequence of SEQ ID NO: 739 and a light chain variableregion comprising the amino acid sequence of SEQ ID NO: 664. The heavychain variable region amino acid sequence of SEQ ID NO: 559 excludes theamino acid sequence of SEQ ID NO: 13. The light chain variable regionamino acid sequence of SEQ ID NO: 664 excludes the amino acid sequenceof SEQ ID NO: 14. In some aspects, an isolated antibody that bindsspecifically to CD38 comprises a heavy chain CDR1 comprising the aminoacid sequence of, SEQ ID NO: 200, SEQ ID NO: 514 or SEQ ID NO: 697, aheavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 202,SEQ ID NO: 516, SEQ ID NO: 544, SEQ ID NO: 698 or SEQ ID NO: 737, and aheavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 204,SEQ ID NO: 222, SEQ ID NO: 518, SEQ ID NO: 534, SEQ ID NO: 535, SEQ IDNO: 536, SEQ ID NO: 699 or SEQ ID NO: 738, and may further comprise alight chain CDR1 comprising the amino acid sequence of SEQ ID NO: 233,SEQ ID NO: 319, SEQ ID NO: 583, SEQ ID NO: 590 or SEQ ID NO: 696, alight chain CDR2 comprising the amino acid sequence of SEQ ID NO: 235,SEQ ID NO: 307, SEQ ID NO: 311, SEQ ID NO: 585, SEQ ID NO: 591 or SEQ IDNO: 605, a light chain CDR3 comprising the amino acid sequence of SEQ IDNO: 237, SEQ ID NO: 321, SEQ ID NO: 324, SEQ ID NO: 587 or SEQ ID NO:594.

In preferred aspects, the heavy chain variable region comprises theamino acid sequence of SEQ ID NO: 34, SEQ ID NO: 18, SEQ ID NO: 665, SEQID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 179, SEQ ID NO:180, SEQ ID NO: 156, SEQ ID NO: 197, SEQ ID NO: 152, SEQ ID NO: 720, SEQID NO: 721, SEQ ID NO: 722, SEQ ID NO: 723, SEQ ID NO: 739, SEQ ID NO:740, SEQ ID NO: 741, SEQ ID NO: 742, SEQ ID NO: 728, SEQ ID NO: 730, SEQID NO: 731. In preferred aspects, the light chain variable regioncomprises the amino acid sequence of SEQ ID NO: 65, SEQ ID NO: 68, SEQID NO: 86, SEQ ID NO: 88, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 660,SEQ ID NO: 661, SEQ ID NO: 662, SEQ ID NO: 663. SEQ ID NO: 161, SEQ IDNO: 184, SEQ ID NO: 185, SEQ ID NO: 188, SEQ ID NO: 198 or SEQ ID NO:700, SEQ ID NO: 701, SEQ ID NO: 704, SEQ ID NO: 705, SEQ ID NO: 706, SEQID NO: 707, SEQ ID NO: 708, SEQ ID NO: 709, SEQ ID NO: 710, SEQ ID NO:711.

The antibody preferably is capable of binding to CD38-positive cells.The antibody may bind to a CD38-positive cell with an EC50 value of lessthan about 100 nM. The antibody may bind to a CD38-positive cell with anEC₅₀ value of less than about 75 nM. The antibody may bind to aCD38-positive cell with an EC₅₀ value of less than about 50 nM. Theantibody may bind to a CD38-positive cell with an EC₅₀ value of lessthan about 30 nM. The antibody may bind to a CD38-positive cell with anEC₅₀ value of less than about 25 nM. The antibody may bind to aCD38-positive cell with an EC₅₀ value of less than about 20 nM. Theantibody may bind to a CD38-positive cell with an EC₅₀ value of lessthan about 15 nM. The antibody may bind to a CD38-positive cell with anEC₅₀ value of less than about 13 nM. The antibody may bind to aCD38-positive cell with an EC₅₀ value of less than about 10 nM.

The antibody may be a monoclonal antibody, and is preferably a fullyhuman antibody. The antibody may comprise an FAb. The antibody maycomprise a human IgG1 constant region or a human IgG4 constant region.The IgG1 or the IgG4 constant region may comprise a tyrosine at position252, a threonine at position 254, and a glutamic acid at position 256according to the EU numbering system. The IgG4 constant region maycomprise a proline at position 228 according to the EU numbering system,and the proline at position 228 may be in addition to a tyrosine atposition 252, a threonine at position 254, and a glutamic acid atposition 256.

In some aspects, the antibody is fused to attenuated interferonalpha-2b. The interferon alpha-2b may comprise a substitution of thealanine at position 145 to glycine or aspartic acid, including aninterferon alpha-2b having the amino acid sequence of SEQ ID NO: 649 orSEQ ID NO: 651. The attenuated interferon alpha-2b may be fused directlyto the C-terminus of the IgG1 or IgG4 constant region, and the antibodymay comprise the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 10, SEQID NO: 652, SEQ ID NO: 653, SEQ ID NO: 654, SEQ ID NO: 655, SEQ ID NO:656, SEQ ID NO: 657, SEQ ID NO: 658, or SEQ ID NO: 694. The antibody,including the antibody fused to an attenuated interferon alpha-2b may becomprised in a composition comprising a pharmaceutically acceptablecarrier.

Isolated polynucleotides encoding the antibody and the antibody fused toan attenuated interferon alpha-2b are provided. The polynucleotide maycomprise the nucleic acid sequence of SEQ ID NO: 667, SEQ ID NO: 670,SEQ ID NO: 671, SEQ ID NO: 672, SEQ ID NO: 673, SEQ ID NO: 674, SEQ IDNO: 668, SEQ ID NO: 669, SEQ ID NO: 675, SEQ ID NO: 676, or SEQ ID NO:677, SEQ ID NO: 678, SEQ ID NO: 679, SEQ ID NO: 680, SEQ ID NO: 681, SEQID NO: 682, SEQ ID NO: 683, SEQ ID NO: 684, SEQ ID NO: 685, SEQ ID NO:686, SEQ ID NO: 687, SEQ ID NO: 688, SEQ ID NO: 689, SEQ ID NO: 690, SEQID NO: 691, SEQ ID NO: 692, SEQ ID NO: 693, SEQ ID NO: 695 SEQ ID NO:702, SEQ ID NO: 703, SEQ ID NO: 712, SEQ ID NO: 713, SEQ ID NO: 714, SEQID NO: 715, SEQ ID NO: 716, SEQ ID NO: 717, SEQ ID NO: 718, SEQ ID NO:719, SEQ ID NO: 724, SEQ ID NO: 725, SEQ ID NO: 726, SEQ ID NO: 727 SEQID NO: 732, SEQ ID NO: 733, SEQ ID NO: 734, SEQ ID NO: 735, SEQ ID NO:743, SEQ ID NO: 744, SEQ ID NO: 745, SEQ ID NO: 746. The polynucleotidesmay comprise a vector. The vector may be used, for example, to transforma cell. A transformed cell comprising such polynucleotides is alsoprovided. The transformed cell may comprise a mammalian cell, a yeastcell, or an insect cell.

Stable cells that express the antibodies are also provided.Antibody-expressing cells may be mammalian cells. Preferred cells areChinese Hamster Ovary (CHO) cells.

Kits comprising antibodies fused to attenuated interferon alpha-2b areprovided. The kits comprise the anti-CD38-attenuated interferon alpha-2bfusion construct, and instructions for using the construct in a methodfor inhibiting the proliferation of a tumor cell expressing CD38 and areceptor for interferon alpha-2b on its surface, instructions for usingthe construct in a method for inducing apoptosis in a tumor cellexpressing CD38 and a receptor for interferon alpha-2b on its surface,instructions for using the construct in a method for treating a tumorcomprising cells expressing CD38 and a receptor for interferon alpha-2bon their surface in a subject in need thereof, and optionally, apharmaceutically acceptable carrier. Kits comprising anti-CD38antibodies are provided, and such kits comprise the anti-CD38 antibodyand instructions for using the antibody in a method for detecting aCD38-positive tumor cell in a tissue sample isolated from a subject, theantibody may optionally be fused to an attenuated interferon alpha-2bprotein.

The anti-CD38 antibody-attenuated interferon alpha-2b fusion constructsmay be used as a therapy in the treatment of a tumor comprising cellsexpressing CD38 and a receptor for interferon alpha-2b on their surface.Generally, a treatment method comprises administering to a subjecthaving the tumor an anti-CD38 antibody-attenuated interferon alpha-2bfusion construct in an amount effective to treat the tumor. Theconstruct may comprise any construct described or exemplified herein.The subject is preferably a mammal, more preferably a non-human primate,and most preferably a human being. The tumor may comprise a B-celllymphoma, multiple myeloma, non-Hodgkin's lymphoma, chronic myelogenousleukemia, chronic lymphocytic leukemia or acute myelogenous leukemia.

The anti-CD38 antibodies, optionally fused to an attenuated interferonalpha-2b protein, may be used in a method for detecting a CD38-positivetumor cell in a tissue sample isolated from a subject. Generally, themethod comprises contacting an antibody that binds specifically to CD38with a tissue sample isolated from a subject and detecting a complex ofthe antibody and a CD38-positive cell in the tissue sample. The tissuesample may be known to have or be suspected of having CD38-positivetumor cells. The tissue may comprise blood or bone marrow. TheCD38-positive tumor cell may be a CD38-positive B-cell lymphoma cell,multiple myeloma cell, non-Hodgkin's lymphoma cell, chronic myelogenousleukemia cell, chronic lymphocytic leukemia cell, or acute myelogenousleukemia cell. The subject is preferably a mammal, more preferably anon-human primate, and most preferably a human being. The method mayinclude the step of isolating the tissue sample from the subject. Themethod may further comprise contacting the antibody with a tissue samplethat does not include any CD38-positive cells, for example, to serve asa negative control.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an example of an anti-CD38-attenuated interferon fusionconstruct.

FIGS. 2A and 2B show sequences of heavy chain variable regions of X02.1,related constructs and the most homologous germline antibody sequence.CDRs defined by the Kabat numbering system are underlined.

FIGS. 3A and 3B show sequences of light chain variable regions of X02.1,related constructs and the most homologous germline antibody sequence.CDRs defined by the Kabat numbering system are underlined.

FIGS. 4A-4D show the sequences of light chain variable regions of A02.1and related constructs. CDRs defined by the Kabat numbering system areunderlined

FIG. 5 shows the consensus variable heavy chain sequence of A02.1 andrelated constructs. Boxed regions contain CDRs (as indicated) as definedby the Kabat numbering system and the enhanced Chothia numbering system.CDRs defined by the Kabat numbering system are shown in bold. CDRsdefined by the enhanced Chothia numbering system are underlined.

FIG. 6 shows the consensus variable light chain sequence of A02.1 andrelated constructs. Boxed regions contain CDRs (as indicated) as definedby the Kabat numbering system and the enhanced Chothia numbering system.CDRs defined by the Kabat numbering system are shown in bold. CDRsdefined by the enhanced Chothia numbering system are underlined

FIGS. 7A-7C show sequences of heavy chain variable regions of humanizedheavy chain variable regions. CDRs defined by the Kabat numbering systemare underlined.

FIGS. 8A-8C show sequences of heavy chain variable regions of humanizedlight chain variable regions. CDRs defined by the Kabat numbering systemare underlined.

FIGS. 9A and 9B show the variable heavy chain of A10.0 and relatedconstructs. CDRs defined by the Kabat numbering system are underlined.

FIGS. 10A and 10B show the variable light chain of A10.0 and relatedconstructs. CDRs defined by the Kabat numbering system are underlined.

FIGS. 11A and 11B show the variable heavy chain consensus sequence ofA10.0 and related constructs. Boxed regions contain CDRs (as indicated)as defined by the Kabat numbering system and the enhanced Chothianumbering system. CDRs defined by the Kabat numbering system are shownin bold. CDRs defined by the enhanced Chothia numbering system areunderlined.

FIG. 12 shows the variable light chain consensus sequence of A10.0 andrelated constructs. Boxed regions contain CDRs (as indicated) as definedby the Kabat numbering system and the enhanced Chothia numbering system.CDRs defined by the Kabat numbering system are shown in bold. CDRsdefined by the enhanced Chothia numbering system are underlined.

FIG. 13 shows the binding activity of A02.1 variants to theCD38-expressing multiple myeloma cell line ARP-1 as measured by flowcytometry. The assay details are described in the Examples of thisspecification.

FIG. 14 shows the binding activity of A02.1 variants to theCD38-expressing multiple myeloma cell line NCI-H929 as measured by flowcytometry. The assay details are described in the Examples of thisspecification.

FIGS. 15 and 16 show the anti-proliferative activity of A02.1 variantson the multiple myeloma cell line ARP-1. A-isotype is an irrelevantspecificity antibody fused with the attenuated interferon as a control.The assay details are described in the Examples (Cell proliferationassay).

FIG. 17 shows the anti-proliferative activity of IFN-alpha2b (Intron A)compared with A02.1 and A10.0 and their corresponding unfused antibodiesX02.1 and X10.0 on the multiple myeloma cell line ARP-1. A-isotype is anirrelevant specificity antibody fused with the attenuated interferon asa control. The assay details are described in the Examples (Cellproliferation assay).

FIG. 18 shows the relative fold change of Annexin V production in theCD38-expressing multiple myeloma cell line NCI-H929 when treated withA02.1 and A10.0 and their corresponding unfused antibodies X02.1 andX10.0 for 24 hours compared to an untreated control. A-isotype is anirrelevant specificity antibody fused with the attenuated interferon asa control. The assay details are described in the Examples (Annexin Vassay).

FIG. 19 shows the relative fold change of caspase activation in theCD38-expressing multiple myeloma cell line H929 of IFN-alpha2b (IntronA) vs. A02.1 and related constructs in comparison to untreated cells.Isotype 145D is an irrelevant specificity antibody fused with theattenuated interferon as a control. The assay details are described inthe Examples (Caspase assay).

FIG. 20 shows the off target activity of IFN-alpha2b (Intron A) versusA02.6 and A02.6 fused to wild-type IFN-alpha2b (A02.6 (wt. IFN)) on theCD38-negative cells. The assay details are described in the Examples(HEK-BLUE™).

FIG. 21 shows the relative fold change of Annexin V production in theCD38-expressing multiple myeloma cell line H929 between IgG1 and IgG4subtypes of anti-CD38-attenuated IFN-alpha fusion protein constructs.A-isotype is a non-specific IgG4 antibody fused with the attenuatedinterferon as a control. The antibodies, A02.12 and A10.0 contain IgG4constant regions fused to attenuated IFN-alpha while A02.112 and A10.59contain IgG1 constant regions fused to attenuated IFN-alpha. The assaydetails are described in the Examples (Annexin V/7AAD assay).

FIG. 22 shows the binding activity of A10.0 variants to theCD38-expressing multiple myeloma cell line NCI-H929 as measured by flowcytometry. The assay details are described in the Examples of thisspecification.

FIG. 23 shows caspase activation in the CD38-expressing multiple myelomacell line H929 of A10.0 and A10.38 compared to untreated cells.A-isotype is an irrelevant specificity antibody fused to the attenuatedIFN as a control. The assay details are described in the Examples(Caspase assay).

FIG. 24 shows the relative fold change of caspase activation in the CD38expressing multiple myeloma cell line H929 by A10.0 variants compared tountreated cells. The assay details are described in the Examples(Caspase assay).

FIG. 25 shows the relative fold change of production of Annexin V in theCD38-expressing multiple myeloma cell line H929 by A10.0 variants. Theassay details are described in the Examples (Annexin V/7AAD assay).

FIG. 26 shows the anti-proliferative activity of IFN-alpha2b (Intron A)compared with A02.6, A10.0, A10.38 and parental A10A2.0 chimericantibody constructs on the Burkitt's lymphoma cell line Daudi. A-isotypeis an irrelevant specificity antibody fused to the attenuated IFN as acontrol. The assay details are described in the Examples (Cellproliferation assay).

FIG. 27 shows the effects of humanized A10.0 versus the parental A10A2.0chimeric antibody attenuated interferon construct on the growth ofsubcutaneous H929 myeloma tumors in SCID mice. The bar labeled“treatment phase” shows the duration of treatment with the compounds.

FIG. 28 shows the non-antibody antigen targeted IFN activity of A10.0variants fused to the same attenuated IFN-alpha2b protein. The assaydetails are described in the Examples (“Off-target assays”—iLite genereporter assay).

FIG. 29 shows the “Off-target” activity of IFN-alpha2b (Intron A)compared with A10.0 variants and the parental A10A2.0 chimeric antibodyfused to wild-type IFN-alpha2b (A10A2.0 chimeric (wt.IFN)). The assaydetails are described in the Examples (“Off-target assays”—HEK-BLUE™).

FIG. 30 shows variable heavy chain consensus sequences ofX910/12-HC-L0-Interferon-alpha (A145D) IgG4 and related sequences. Boxedregions contain CDRs (as indicated) as defined by the Kabat numberingsystem and the enhanced Chothia numbering system. CDRs defined by theKabat numbering system are shown in bold. CDRs defined by the enhancedChothia numbering system are underlined.

DETAILED DESCRIPTION OF THE DISCLOSURE

Various terms relating to aspects of disclosure are used throughout thespecification and claims. Such terms are to be given their ordinarymeaning in the art, unless otherwise indicated. Other specificallydefined terms are to be construed in a manner consistent with thedefinition provided herein.

The terms subject and patient are used interchangeably and include anyanimal. Mammals are preferred, including companion and farm mammals, aswell as rodents, including mice, rabbits, and rats, and other rodents.Non-human primates, such as Cynomolgus monkeys, are more preferred, andhuman beings are highly preferred.

A molecule such as an antibody has been “isolated” if it has beenaltered and/or removed from its natural environment by the hand of ahuman being.

As used herein, the singular forms “a,” “an,” and “the” include pluralreferents unless expressly stated otherwise.

An anti-CD38 antibody-attenuated interferon alpha-2b fusion constructincludes, but is not limited to, any antibody described or exemplifiedherein that binds specifically to CD38 that is fused to an attenuatedinterferon alpha-2b protein, including an interferon alpha-2b of SEQ IDNO: 647, SEQ ID NO: 648, SEQ ID NO: 649, SEQ ID NO: 650, or SEQ ID NO:651. In some aspects, fusing an unmutated interferon alpha-2b protein,such as SEQ ID NO: 7, to an anti-CD38 antibody attenuates the biologicactivities of the interferon molecule. In this disclosure, attenuatedinterferon, attenuated interferon alpha-2b, IFN-alpha2b A145D, andIFN-alpha2b A145G are used interchangeably.

Specificity is not necessarily an absolute designation but mayconstitute a relative term signifying the degree of selectivity of anantibody IFN-alpha fusion protein construct for an antigen-positive cellcompared to an antigen-negative cell. Specificity of an antibodyIFN-alpha fusion protein construct for an antigen-postive cell ismediated by the variable regions of the antibody, and usually by thecomplementarity determining regions (CDRs) of the antibody. A constructmay have 100-fold specificity for antigen-positive cells compared toantigen-negative cells.

Human CD38 comprises the amino acid sequence of SEQ ID NO: 1, andcynomolgus monkey CD38 comprises the amino acid sequence of SEQ ID NO:2.

It has been further observed that interferon-alpha2b can be attenuatedin terms of its biologic activity which is mediated through theinterferon binding to an interferon receptor on a cell surface byintroducing certain amino acid changes into the protein sequence. Anattenuated interferon molecule can be fused to antibodies thatspecifically bind to CD38, such that the antibody may serve as adelivery vehicle for the attenuated interferon to CD38-positive cellswith a resulting diminution of off target interferon activity caused bythe attenuated interferon molecule. It has been further observed thatfusing the attenuated interferon to the CD38 antibodies does notsignificantly affect the capacity of the antibody to specifically bindto CD38 on cells expressing CD38, including cells in the body ofanimals. It has been further observed that variants of the CD38antibodies can be engineered and expressed such that the antibodies havereduced immunogenicity and enhanced stability and half life without asignificant loss of specificity or affinity of the antibody to the CD38antigen. These variant antibodies can be fused to an attenuatedinterferon.

Accordingly, antibodies that specifically bind to CD38 are featured. Ithas also been observed that such anti-CD38 antibodies may be employed asdelivery vehicles for attenuated ligands such as interferon alpha.Without intending to be limited to any particular theory or mechanism ofaction, it is believed that the antibodies direct the interferon alphato which they are attached to CD38-positive cells, where the interferonmay interact with its receptor. It is believed that the antibody, as adelivery vehicle, compensates for the diminished capacity of theinterferon molecule to bind to its receptor. In this sense, theattenuated interferon has reduced capacity to interact with its receptoron healthy cells, and particularly cells that do not express CD38. It isbelieved that by bringing the attenuated interferon into proximity withits receptor on CD38-positive cells, the antibodies may enhance thecapacity of the attenuated interferon to bind to its relevant receptorand induce a therapeutic effect, while exhibiting a diminished capacityto induce undesirable effects on healthy cells that do not express CD38.

The antibodies may be polyclonal, but in some aspects, are notpolyclonal. The antibodies preferably are monoclonal. The antibodies arepreferably full length antibodies. Full length antibodies generallycomprise a variable region heavy chain and a variable region lightchain. The antibodies may comprise derivatives or fragments or portionsof antibodies that retain the antigen-binding specificity, and alsopreferably retain most or all of the affinity, of the parent antibodymolecule (e.g., for CD38). For example, derivatives may comprise atleast one variable region (either a heavy chain or light chain variableregion). Other examples of suitable antibody derivatives and fragmentsinclude, without limitation, antibodies with polyepitopic specificity,bispecific antibodies, multi-specific antibodies, diabodies,single-chain molecules, as well as FAb, F(Ab′)2, Fd, Fabc, and Fvmolecules, single chain (Sc) antibodies, single chain Fv antibodies(scFv), individual antibody light chains, individual antibody heavychains, fusions between antibody chains and other molecules, heavy chainmonomers or dimers, light chain monomers or dimers, dimers consisting ofone heavy and one light chain, and other multimers. Single chain Fvantibodies may be multi-valent. All antibody isotypes may be used toproduce antibody derivatives, fragments, and portions. Antibodyderivatives, fragments, and/or portions may be recombinantly producedand expressed by any cell type, prokaryotic or eukaryotic.

In some embodiments an isolated antibody may refer to a monoclonalantibody to which IFN-alpha, or an attenuated IFN-alpha, has been fusedto the C-terminus of the heavy chain IgG constant region. When themonoclonal antibody has a binding specificity to CD38 and the IFN-alphais attenuated IFN-alpha 2b, the isolated antibody is also referred to asan Anti-CD38 attenuated IFN-alpha fusion protein, or an Anti-CD38attenutated IFN-alpha fusion construct herein.

In a full-length antibody, each heavy chain is comprised of a heavychain variable region (abbreviated herein as HCVR or VH) and a heavychain constant region. The heavy chain constant region is comprised ofthree domains, CH1, CH2 and CH3. Each light chain is comprised of alight chain variable region (abbreviated herein as LCVR or VL) and alight chain constant region. The light chain constant region iscomprised of one domain, CL. The VH and VL regions can be furthersubdivided into regions of hypervariability, termed complementaritydetermining regions (CDR), interspersed with regions that are moreconserved, termed framework regions (FWR or FR). Each VH and VL iscomposed of three CDRs and four FWRs, arranged from amino-terminus tocarboxy-terminus in the following order: FWR1, CDR1, FWR2, CDR2, FWR3,CDR3, FWR4. Typically, the antigen binding properties of an antibody areless likely to be disturbed by changes to FWR sequences than by changesto the CDR sequences. Immunoglobulin molecules can be of any type (e.g.,IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4,IgA1 and IgA2) or subclass.

The antibodies may be derived from any species. For example, theantibodies may be mouse, rat, goat, horse, swine, bovine, camel,chicken, rabbit, donkey, llama, dromedary, shark, or human antibodies,as well as antibodies from any other animal species. For use in thetreatment of humans, non-human derived antibodies may be structurallyaltered to be less antigenic upon administration to a human patient,including by chimerization or humanization or superhumanization.

In some aspects, the antibodies are humanized antibodies. Humanizedantibodies are those wherein the amino acids directly involved inantigen binding, e.g., the complementarity determining regions (CDR),and in some cases the framework regions (FWR), or portions thereof, ofthe heavy and/or light chains are not of human origin, while the rest ofthe amino acids in the antibody are human or otherwise of human origin,e.g., a human antibody scaffold. Humanized antibodies also includeantibodies in which one or more residues of the human protein aremodified by one or more amino acid substitutions and/or one or more FWRresidues of the human protein are replaced by corresponding non-humanresidues. Humanized antibodies may also comprise residues which arefound in neither the human antibody or in the non-human antibody. Ahumanized antibody may be a super-humanized antibody, e.g., as describedin U.S. Pat. No. 7,732,578. The antibodies may be humanized chimericantibodies.

In highly preferred aspects, the antibodies are fully human. Fully humanantibodies are those where the whole molecule is human or otherwise ofhuman origin, or includes an amino acid sequence identical to a humanform of the antibody. Fully human antibodies include those obtained froma human V gene library, for example, where human genes encoding variableregions of antibodies are recombinantly expressed. Fully humanantibodies may be expressed in other organisms (e.g., mice and xenomousetechnology) or cells from other organisms transformed with genesencoding human antibodies. Fully human antibodies may neverthelessinclude amino acid residues not encoded by human sequences, e.g.,mutations introduced by random or site directed mutations.

The antibodies may be full length antibodies of any class, for example,IgG1, IgG2 or IgG4. The constant domains of such antibodies arepreferably human. The variable regions of such antibodies may be ofnon-human origin, or preferably are human in origin or are humanized.Antibody fragments may also be used in place of the full lengthantibodies.

The antibodies may be minibodies. Minibodies comprise small versions ofwhole antibodies, which encode in a single chain the essential elementsof a whole antibody. For example, the minibody may be comprised of theVH and VL domains of a native antibody fused to the hinge region and CH3domain of an immunoglobulin molecule.

In some aspects, the antibody may comprise non-immunoglobulin derivedprotein frameworks. For example, reference may be made to (Ku & Schutz,Proc. Natl. Acad. Sci. USA 92: 6552-6556, 1995) which describes afour-helix bundle protein cytochrome b562 having two loops randomized tocreate CDRs, which have been selected for antigen binding.

Natural sequence variations may exist among heavy and light chains andthe genes encoding them, and therefore, persons having ordinary skill inthe art would expect to find some level of variation within the aminoacid sequences, or the genes encoding them, of the antibodies describedand exemplified herein. These variants preferably maintain the uniquebinding properties (e.g., specificity and affinity) of the parentantibody. Such an expectation is due in part to the degeneracy of thegenetic code, as well as to the known evolutionary success ofconservative amino acid sequence variations, which do not appreciablyalter the nature of the encoded protein. Accordingly, such variants andhomologs are considered substantially the same as one another and areincluded within the scope of the disclosure. The antibodies thus includevariants having single or multiple amino acid substitutions, deletions,additions, or replacements that retain the biological properties (e.g.,binding specificity and binding affinity) of the parent antibodies. Thevariants are preferably conservative, but may be non-conservative.

Amino acid positions assigned to CDRs and FWRs may be defined accordingto Kabat Sequences of Proteins of Immunological Interest, NationalInstitutes of Health, Bethesda, Md., 1987 and 1991 (also referred toherein as the Kabat numbering system). In addition, the amino acidpositions assigned to CDRs and FWRs may be defined according to theEnhanced Chothia Numbering Scheme (http://www.bioinfo.org.uk/mdex.html).The heavy chain constant region of an antibody can be defined by the EUnumbering system (Edelman, G M et al. (1969)., Proc. Natl. Acad. USA,63, 78-85).

According to the numbering system of Kabat, VH FWRs and CDRs may bepositioned as follows: residues 1-30 (FWR1), 31-35 (CDR1), 36-49 (FWR2),50-65 (CDR2), 66-94 (FWR3), 95-102 (CDR3) and 103-113 (FWR4), and VLFWRs and CDRs are positioned as follows: residues 1-23 (FWR1), 24-34(CDR1), 35-49 (FWR2), 50-56 (CDR2), 57-88 (FWR3), 89-97 (CDR3) and98-107 (FWR4). In some instances, variable regions may increase inlength and according to the Kabat numbering system some amino acids maybe designated by a number followed by a letter. This specification isnot limited to FWRs and CDRs as defined by the Kabat numbering system,but includes all numbering systems, including the canonical numberingsystem or of Chothia et al. (1987) J. Mol Biol. 196:901-17; Chothia etal. (1989) Nature 342:877-83; and/or Al-Lazikani et al. (1997) J. Mol.Biol. 273:927-48; the numbering system of Honnegher et al. (2001) J.Mol. Biol., 309:657-70; or the IMGT system discussed in Giudicelli etal., (1997) Nucleic Acids Res. 25:206-11. In some aspects, the CDRs aredefined according to the Kabat numbering system.

In some particular aspects, for any of the heavy chain CDR2 subdomainsdescribed herein, according to the Kabat numbering system, the fiveC-terminal amino acids may not participate directly in antigen binding,and accordingly, it will be understood that any one or more of thesefive C-terminal amino acids may be substituted with anothernaturally-occurring amino acid without substantially adversely affectingantigen binding. In some aspects, for any of the light chain CDR1subdomains described herein, according to the Kabat numbering system,the four N-terminal amino acids may not participate directly in antigenbinding, and accordingly, it will be understood that any one or more ofthese four amino acids may be substituted with anothernaturally-occurring amino acid without substantially adversely affectingantigen binding. For example, as described by Padlan et al. (1995) FASEBJ. 9:133-139, the five C terminal amino acids of heavy chain CDR2 and/orthe four N-terminal amino acids of light chain CDR1 may not participatein antigen binding. In some aspects, both the heavy chain CDR2 and thelight chain CDR1 do not directly participate in antigen binding.

In some aspects, chemical analogues of amino acids may be used in theantibodies described and/or exemplified herein. The use of chemicalanalogues of amino acids is useful, for example, for stabilizing themolecules such as if required to be administered to a subject. Theanalogues of the amino acids contemplated herein include, but are notlimited to, modifications of side chains, incorporation of unnaturalamino acids and/or their derivatives during peptide, polypeptide orprotein synthesis and the use of crosslinkers and other methods whichimpose conformational constraints on the proteinaceous molecule or theiranalogues.

The antibodies may comprise post-translational modifications ormoieties, which may impact antibody activity or stability. Thesemodifications or moieties include, but are not limited to, methylated,acetylated, glycosylated, sulfated, phosphorylated, carboxylated, andamidated moieties and other moieties that are well known in the art.Moieties include any chemical group or combinations of groups commonlyfound on immunoglobulin molecules in nature or otherwise added toantibodies by recombinant expression systems, including prokaryotic andeukaryotic expression systems.

Examples of side chain modifications contemplated by the disclosureinclude modifications of amino groups such as by reductive alkylation byreaction with an aldehyde followed by reduction with NaBH₄; amidinationwith methylacetimidate; acylation with acetic anhydride; carbamoylationof amino groups with cyanate; trinitrobenzylation of amino groups with2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groupswith succinic anhydride and tetrahydrophthalic anhydride; andpyridoxylation of lysine with pyridoxal-5-phosphate followed byreduction with NaBH₄.

The guanidine group of arginine residues may be modified by theformation of heterocyclic condensation products with reagents such as2,3-butanedione, phenylglyoxal and glyoxal.

The carboxyl group may be modified by carbodiimide activation viaO-acylisourea formation followed by subsequent derivation, for example,to a corresponding amide.

Sulphydryl groups may be modified by methods such as carboxymethylationwith iodoacetic acid or iodoacetamide; performic acid oxidation tocysteic acid; formation of mixed disulphides with other thiol compounds;reaction with maleimide, maleic anhydride or other substitutedmaleimide; formation of mercurial derivatives using4-chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid,phenylmercury chloride, 2-chloromercuri-4-nitrophenol and othermercurials; carbamoylation with cyanate at alkaline pH.

Tryptophan residues may be modified by, for example, oxidation withN-bromosuccinimide or alkylation of the indole ring with2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine residueson the other hand, may be altered by nitration with tetranitromethane toform a 3-nitrotyrosine derivative.

Modification of the imidazole ring of a histidine residue may beaccomplished by alkylation with iodoacetic acid derivatives orN-carbethoxylation with diethylpyrocarbonate.

Crosslinkers may be used, for example, to stabilize 3D conformations ofthe antibodies and constructs, using homo-bifunctional crosslinkers suchas the bifunctionalimido esters having (CH₂)n spacer groups with n=1 ton=6, glutaraldehyde, N-hydroxysuccinimide esters and hetero-bifunctionalreagents which usually contain an amino-reactive moiety such asN-hydroxysuccinimide and another group specific-reactive moiety such asmaleimido or dithio moiety (SH) or carbodiimide (COOH).

The antibodies may be affinity matured, or may comprise amino acidchanges that decrease immunogenicity, for example, by removing predictedMHC class II-binding motifs. The therapeutic utility of the antibodiesdescribed herein may be further enhanced by modulating their functionalcharacteristics, such as antibody-dependent cell-mediated cytotoxicity(ADCC), complement-dependent cytotoxicity (CDC), serum half-life,biodistribution and binding to Fc receptors or the combination of any ofthese. This modulation can be achieved by protein-engineering,glyco-engineering or chemical methods. Depending on the therapeuticapplication required, it could be advantageous to either increase ordecrease any of these activities. An example of glyco-engineering usedthe Potelligent® method as described in Shinkawa T. et al. (2003) J.Biol. Chem. 278: 3466-73.

The antibodies may include modifications that modulate its serumhalf-life and biodistribution, including modifications that modulate theantibody's interaction with the neonatal Fc receptor (FcRn), a receptorwith a key role in protecting IgG from catabolism, and maintaining highserum antibody concentration. Serum half-life modulating modificationsmay occur in the Fc region of IgG1 or IgG4, including the triplesubstitution of M252Y/S254T/T256E (Numbering according to the EUnumbering system (Edelman, G. M. et al. (1969) Proc. Natl. Acad. USA 63,78-85)), (e.g., SEQ ID NO: 656, SEQ ID NO: 657, SEQ ID NO: 658, SEQ IDNO: 694), as described in U.S. Pat. No. 7,083,784. Other substitutionsmay occur at positions 250 and 428, see e.g., U.S. Pat. No. 7,217,797,as well as at positions 307, 380 and 434, see, e.g., WO 00/42072.Examples of constant domain amino acid substitutions which modulatebinding to Fc receptors and subsequent function mediated by thesereceptors, including FcRn binding and serum half-life, are described inU.S. Publ. Nos. 2009/0142340, 2009/0068175, and 2009/0092599. Nakedantibodies may have the heavy chain C-terminal lysine omitted or removedto reduce heterogeneity. The substitution of S228P (EU numbering) in thehuman IgG4 can stabilize antibody Fab-arm exchange in vivo (Labrin etal. (2009) Nature Biotechnology 27:8; 767-773).

The glycans linked to antibody molecules are known to influenceinteractions of antibody with Fc receptors and glycan receptors andthereby influence antibody activity, including serum half-life. Hence,certain glycoforms that modulate desired antibody activities can confertherapeutic advantage. Methods for generating engineered glycoformsinclude but are not limited to those described in U.S. Pat. Nos.6,602,684, 7,326,681, and 7,388,081 and PCT Publ. No. WO 08/006554.Alternatively, the antibody sequences may be modified to remove relevantglycoform attachment sites.

The antibodies may be labeled or conjugated to any chemical orbiomolecule moieties. Labeled antibodies may find use in therapeutic,diagnostic, or basic research applications. Such labels/conjugates canbe detectable, such as fluorochromes, radiolabels, enzymes, fluorescentproteins, and biotin. The labels/conjugates may be chemotherapeuticagents, toxins, isotopes, and other agents used for treating conditionssuch as the killing of cancer cells. Chemotherapeutic agents may be anywhich is suitable for the purpose to which the antibody is being used.

The antibodies may be derivatized by known protecting/blocking groups toprevent proteolytic cleavage or enhance activity or stability.

The antibodies preferably have a binding affinity for an epitope on CD38that includes a dissociation constant (Kd) of less than about 1×10⁻² M.In some embodiments, the Kd is less than about 1×10⁻³ M. In otherembodiments, the Kd is less than about 1×10⁻⁴M. In some embodiments, theKd is less than about 1×10^(−s) M. In still other embodiments, the Kd isless than about 1×10⁻⁶ M. In other embodiments, the Kd is less thanabout 1×10⁻⁷M. In other embodiments, the Kd is less than about 1×10⁻⁸ M.In other embodiments, the Kd is less than about 1×10⁻⁹ M. In otherembodiments, the Kd is less than about 1×10⁻¹⁰ M. In still otherembodiments, the Kd is less than about 1×10⁻¹¹ M. In some embodiments,the Kd is less than about 1×10⁻¹² M. In other embodiments, the Kd isless than about 1×10⁻¹³M. In other embodiments, the Kd is less thanabout 1×10⁻¹⁴ M. In still other embodiments, the Kd is less than about1×10⁻¹⁵ M. Affinity values refer to those obtained by standardmethodologies, including surface plasmon resonance such as Biacore™analyses or analysis using an Octet® Red 96 (Forte Bio) Dip-and-Readsystem.

The antibodies may comprise a single chain Fv molecule (scFv), Fab, orfull IgG. Any such antibodies may comprise a heavy chain having an aminoacid sequence having at least about 85%, at least about 90%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,at least about 99%, or at least about 100% sequence identity with theamino acid sequence of SEQ ID NO: 659 or SEQ ID NO: 665 or SEQ ID NO:736, provided that a heavy chain comprising the amino acid sequence ofSEQ ID NO: 659 or variant thereof excludes the amino acid sequence ofSEQ ID NO: 13. It will be understood that antibodies comprising aminoacid changes in their heavy chain retain the capability to specificallybind to CD38. The retained CD38 specific binding activity (includingaffinity) is preferably about the same as the binding activity(including affinity) of an antibody without any amino acid changes inthe heavy chain, although the binding activity (including affinity) maybe lesser or greater than an antibody without any amino acid changes inthe heavy chain. The antibody may comprise a light chain having an aminoacid sequence having at least about 85%, at least about 90%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,at least about 99%, or at least about 100% sequence identity with theamino acid sequence of SEQ ID NO: 664 or SEQ ID NO: 666, provided that alight chain comprising the amino acid sequence of SEQ ID NO: 664 orvariant thereof excludes the amino acid sequence of SEQ ID NO: 14. Itwill be understood that antibodies comprising amino acid changes intheir light chain retain the capability to specifically bind to CD38.The retained CD38 specific binding activity (including affinity) ispreferably about the same as the binding activity (including affinity)of an antibody without any amino acid changes in the light chain,although the binding activity (including affinity) may be lesser orgreater than an antibody without any amino acid changes in the lightchain.

In some aspects, the heavy chain FWR1 comprises the amino acid sequenceof SEQ ID NO: 199, SEQ ID NO: 206, SEQ ID NO: 214, SEQ ID NO: 215, SEQID NO: 217, SEQ ID NO: 219, SEQ ID NO: 389, SEQ ID NO: 396, SEQ ID NO:400, SEQ ID NO: 404, SEQ ID NO: 408, SEQ ID NO: 412, SEQ ID NO: 416, SEQID NO: 420, SEQ ID NO: 424, SEQ ID NO: 428, SEQ ID NO: 432, SEQ ID NO:466, SEQ ID NO: 470, SEQ ID NO: 472, SEQ ID NO: 474, SEQ ID NO: 476, SEQID NO: 478, SEQ ID NO: 480, SEQ ID NO: 482, SEQ ID NO: 486, SEQ ID NO:488, SEQ ID NO: 513, SEQ ID NO: 537, SEQ ID NO: 542, SEQ ID NO: 547, SEQID NO: 552, SEQ ID NO: 557, SEQ ID NO: 562, SEQ ID NO: 567, SEQ ID NO:572, SEQ ID NO: 577 or SEQ ID NO: 748, and in some aspects, the heavychain FWR1 comprises an amino acid sequence having at least about 85%,at least about 90%, at least about 92%, at least about 93%, at leastabout 94%, at least about 95%, at least about 96%, at least about 97%,at least about 98%, or at least about 99% sequence identity with theamino acid sequence of SEQ ID NO: 199, SEQ ID NO: 214, SEQ ID NO: 215,SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO: 389, SEQ ID NO: 396, SEQ IDNO: 400, SEQ ID NO: 404, SEQ ID NO: 408, SEQ ID NO: 412, SEQ ID NO: 416,SEQ ID NO: 420, SEQ ID NO: 424, SEQ ID NO: 428, SEQ ID NO: 432, SEQ IDNO: 466, SEQ ID NO: 470, SEQ ID NO: 472, SEQ ID NO: 474, SEQ ID NO: 476,SEQ ID NO: 478, SEQ ID NO: 480, SEQ ID NO: 482, SEQ ID NO: 486, SEQ IDNO: 488, SEQ ID NO: 513, SEQ ID NO: 537, SEQ ID NO: 542, SEQ ID NO: 547,SEQ ID NO: 552, SEQ ID NO: 557, SEQ ID NO: 562, SEQ ID NO: 567, SEQ IDNO: 572, SEQ ID NO: 577 or SEQ ID NO: 748. In some aspects, the heavychain FWR2 comprises the amino acid sequence of SEQ ID NO: 201, SEQ IDNO: 211, SEQ ID NO: 229, SEQ ID NO: 391, SEQ ID NO: 397, SEQ ID NO: 401,SEQ ID NO: 405, SEQ ID NO: 409, SEQ ID NO: 413, SEQ ID NO: 417, SEQ IDNO: 421, SEQ ID NO: 425, SEQ ID NO: 429, SEQ ID NO: 433, SEQ ID NO: 515,SEQ ID NO: 520, SEQ ID NO: 521, SEQ ID NO: 522, SEQ ID NO: 523, SEQ IDNO: 524, SEQ ID NO: 525, SEQ ID NO: 538, SEQ ID NO: 543, SEQ ID NO: 548,SEQ ID NO: 553, SEQ ID NO: 558, SEQ ID NO: 563, SEQ ID NO: 568, SEQ IDNO: 573, SEQ ID NO: 578, SEQ ID NO: 749 or SEQ ID NO: 750, and in someaspects, the heavy chain FWR2 comprises an amino acid sequence having atleast about 85%, at least about 90%, at least about 92%, at least about93%, at least about 94%, at least about 95%, at least about 96%, atleast about 97%, at least about 98%, or at least about 99% sequenceidentity with the amino acid sequence of SEQ ID NO: 201, SEQ ID NO: 211,SEQ ID NO: 229, SEQ ID NO: 391, SEQ ID NO: 397, SEQ ID NO: 401, SEQ IDNO: 405, SEQ ID NO: 409, SEQ ID NO: 413, SEQ ID NO: 417, SEQ ID NO: 421,SEQ ID NO: 425, SEQ ID NO: 429, SEQ ID NO: 433, SEQ ID NO: 515, SEQ IDNO: 520, SEQ ID NO: 521, SEQ ID NO: 522, SEQ ID NO: 523, SEQ ID NO: 524,SEQ ID NO: 525, SEQ ID NO: 538, SEQ ID NO: 543, SEQ ID NO: 548, SEQ IDNO: 553, SEQ ID NO: 558, SEQ ID NO: 563, SEQ ID NO: 568, SEQ ID NO: 573,SEQ ID NO: 578, SEQ ID NO: 749 or SEQ ID NO: 750. In some aspects, theheavy chain FWR3 comprises the amino acid sequence of SEQ ID NO: 203,SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 216, SEQ IDNO: 218, SEQ ID NO: 221, SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 230,SEQ ID NO: 393, SEQ ID NO: 399, SEQ ID NO: 403, SEQ ID NO: 407, SEQ IDNO: 411, SEQ ID NO: 415, SEQ ID NO: 419, SEQ ID NO: 423, SEQ ID NO: 427,SEQ ID NO: 431, SEQ ID NO: 435, SEQ ID NO: 468, SEQ ID NO: 517, SEQ IDNO: 530, SEQ ID NO: 531, SEQ ID NO: 532, SEQ ID NO: 533, SEQ ID NO: 540,SEQ ID NO: 545, SEQ ID NO: 550, SEQ ID NO: 555, SEQ ID NO: 560, SEQ IDNO: 565, SEQ ID NO: 570, SEQ ID NO: 575, SEQ ID NO: 580, SEQ ID NO: 751or SEQ ID NO: 752 and in some aspects, the heavy chain FWR3 comprises anamino acid sequence having at least about 85%, at least about 90%, atleast about 92%, at least about 93%, at least about 94%, at least about95%, at least about 96%, at least about 97%, at least about 98%, or atleast about 99% sequence identity with the amino acid sequence of SEQ IDNO: 203, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 216,SEQ ID NO: 218, SEQ ID NO: 221, SEQ ID NO: 226, SEQ ID NO: 227, SEQ IDNO: 230, SEQ ID NO: 393, SEQ ID NO: 399, SEQ ID NO: 403, SEQ ID NO: 407,SEQ ID NO: 411, SEQ ID NO: 415, SEQ ID NO: 419, SEQ ID NO: 423, SEQ IDNO: 427, SEQ ID NO: 431, SEQ ID NO: 435, SEQ ID NO: 468, SEQ ID NO: 517,SEQ ID NO: 530, SEQ ID NO: 531, SEQ ID NO: 532, SEQ ID NO: 533, SEQ IDNO: 540, SEQ ID NO: 545, SEQ ID NO: 550, SEQ ID NO: 555, SEQ ID NO: 560,SEQ ID NO: 565, SEQ ID NO: 570, SEQ ID NO: 575, SEQ ID NO: 580, SEQ IDNO: 751 or SEQ ID NO: 752. In some aspects, the heavy chain FWR4comprises the amino acid sequence of SEQ ID NO: 205, SEQ ID NO: 395, SEQID NO: 519, SEQ ID NO: 541, SEQ ID NO: 546, SEQ ID NO: 551, SEQ ID NO:556, SEQ ID NO: 561, SEQ ID NO: 566, SEQ ID NO: 571, SEQ ID NO: 576, SEQID NO: 581 or SEQ ID NO: 753, and in some aspects, the heavy chain FWR4comprises an amino acid sequence having at least about 85%, at leastabout 90%, at least about 92%, at least about 93%, at least about 94%,at least about 95%, at least about 96%, at least about 97%, at leastabout 98%, or at least about 99% sequence identity with the amino acidsequence of SEQ ID NO: 205, SEQ ID NO: 395, SEQ ID NO: 519, SEQ ID NO:541, SEQ ID NO: 546, SEQ ID NO: 551, SEQ ID NO: 556, SEQ ID NO: 561, SEQID NO: 566, SEQ ID NO: 571, SEQ ID NO: 576, SEQ ID NO: 581 or SEQ ID NO:753. It will be understood that antibodies comprising amino acid changesin the heavy chain framework region(s) (FWR1, FWR2, FWR3, and/or FWR4)retain the capability to specifically bind to CD38. The retained CD38specific binding activity (including affinity) is preferably about thesame as the binding activity (including affinity) of an antibody withoutany amino acid changes in any heavy chain framework region(s), althoughthe binding activity (including affinity) may be lesser or greater thanan antibody without any amino acid changes in any heavy chain frameworkregion(s).

In some aspects, the heavy chain CDR1 comprises the amino acid sequenceof SEQ ID NO: 200, SEQ ID NO: 224, SEQ ID NO: 390, SEQ ID NO: 514, SEQID NO: 526, SEQ ID NO: 527, SEQ ID NO: 528, SEQ ID NO: 529, or SEQ IDNO: 697 and in some aspects, the heavy chain CDR1 comprises an aminoacid sequence having at least about 85%, at least about 90%, at leastabout 92%, at least about 93%, at least about 94%, at least about 95%,at least about 96%, at least about 97%, at least about 98%, or at leastabout 99% sequence identity with the amino acid sequence of SEQ ID NO:200, SEQ ID NO: 224, SEQ ID NO: 390, SEQ ID NO: 514, SEQ ID NO: 526, SEQID NO: 527, SEQ ID NO: 528, SEQ ID NO: 529, or SEQ ID NO: 697. In someaspects, the heavy chain CDR2 comprises the amino acid sequence of SEQID NO: 202, SEQ ID NO: 392, SEQ ID NO: 398, SEQ ID NO: 402, SEQ ID NO:406, SEQ ID NO: 410, SEQ ID NO: 414, SEQ ID NO: 418, SEQ ID NO: 422, SEQID NO: 426, SEQ ID NO: 430, SEQ ID NO: 434, SEQ ID NO: 467, SEQ ID NO:471, SEQ ID NO: 473, SEQ ID NO: 475, SEQ ID NO: 477, SEQ ID NO: 479, SEQID NO: 481, SEQ ID NO: 483, SEQ ID NO: 485, SEQ ID NO: 487, SEQ ID NO:489, SEQ ID NO: 516, SEQ ID NO: 539, SEQ ID NO: 544, SEQ ID NO: 549, SEQID NO: 554, SEQ ID NO: 559, SEQ ID NO: 564, SEQ ID NO: 569, SEQ ID NO:574, SEQ ID NO: 579, SEQ ID NO: 698 or SEQ ID NO: 737 and in someaspects, the heavy chain CDR2 comprises an amino acid sequence having atleast about 85%, at least about 90%, at least about 92%, at least about93%, at least about 94%, at least about 95%, at least about 96%, atleast about 97%, at least about 98%, or at least about 99% sequenceidentity with the amino acid sequence of SEQ ID NO: 202, SEQ ID NO: 392,SEQ ID NO: 398, SEQ ID NO: 402, SEQ ID NO: 406, SEQ ID NO: 410, SEQ IDNO: 414, SEQ ID NO: 418, SEQ ID NO: 422, SEQ ID NO: 426, SEQ ID NO: 430,SEQ ID NO: 434, SEQ ID NO: 467, SEQ ID NO: 471, SEQ ID NO: 473, SEQ IDNO: 475, SEQ ID NO: 477, SEQ ID NO: 479, SEQ ID NO: 481, SEQ ID NO: 483,SEQ ID NO: 485, SEQ ID NO: 487, SEQ ID NO: 489, SEQ ID NO: 516, SEQ IDNO: 539, SEQ ID NO: 544, SEQ ID NO: 549, SEQ ID NO: 554, SEQ ID NO: 559,SEQ ID NO: 564, SEQ ID NO: 569, SEQ ID NO: 574, SEQ ID NO: 579, SEQ IDNO: 698 or SEQ ID NO: 737. In some aspects, the heavy chain CDR3comprises the amino acid sequence of SEQ ID NO: 204, SEQ ID NO: 220, SEQID NO: 222, SEQ ID NO: 223, SEQ ID NO: 228, SEQ ID NO: 231, SEQ ID NO:394, SEQ ID NO: 469, SEQ ID NO: 518, SEQ ID NO: 534, SEQ ID NO: 535, SEQID NO: 536, SEQ ID NO: 699 or SEQ ID NO: 738 and in some aspects, theheavy chain CDR3 comprises an amino acid sequence having at least about85%, at least about 90%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99% sequence identity withthe amino acid sequence of SEQ ID NO: 204, SEQ ID NO: 220, SEQ ID NO:222, SEQ ID NO: 223, SEQ ID NO: 228, SEQ ID NO: 231, SEQ ID NO: 394, SEQID NO: 469, SEQ ID NO: 518, SEQ ID NO: 534, SEQ ID NO: 535, SEQ ID NO:536, SEQ ID NO: 699 or SEQ ID NO: 738. It will be understood thatantibodies comprising amino acid changes in the heavy chaincomplementarity determining region(s) (CDR1, CDR2, and/or CDR3) retainthe capability to specifically bind to CD38. The retained CD38 specificbinding activity (including affinity) is preferably about the same asthe binding activity (including affinity) of an antibody without anyamino acid changes in any heavy chain complementarity determiningregion(s), although the binding activity (including affinity) may belesser or greater than an antibody without any amino acid changes in anyheavy chain complementarity determining region(s).

In some aspects, the light chain FWR1 comprises the amino acid sequenceof SEQ ID NO: 232, SEQ ID NO: 247, SEQ ID NO: 259, SEQ ID NO: 260, SEQID NO: 261, SEQ ID NO: 436, SEQ ID NO: 443, SEQ ID NO: 447, SEQ ID NO:451, SEQ ID NO: 455, SEQ ID NO: 459, SEQ ID NO: 463, SEQ ID NO: 490, SEQID NO: 497, SEQ ID NO: 501, SEQ ID NO: 509, SEQ ID NO: 582, SEQ ID NO:607, SEQ ID NO: 614, SEQ ID NO: 618, SEQ ID NO: 622, SEQ ID NO: 626, SEQID NO: 630, SEQ ID NO: 634 or SEQ ID NO: 638 and in some aspects, thelight chain FWR1 comprises an amino acid sequence having at least about85%, at least about 90%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99% sequence identity withthe amino acid sequence of SEQ ID NO: 232, SEQ ID NO: 247, SEQ ID NO:259, SEQ ID NO: 260, SEQ ID NO: 261, SEQ ID NO: 436, SEQ ID NO: 443, SEQID NO: 447, SEQ ID NO: 451, SEQ ID NO: 455, SEQ ID NO: 459, SEQ ID NO:463, SEQ ID NO: 490, SEQ ID NO: 497, SEQ ID NO: 501, SEQ ID NO: 509, SEQID NO: 582, SEQ ID NO: 607, SEQ ID NO: 614, SEQ ID NO: 618, SEQ ID NO:622, SEQ ID NO: 626, SEQ ID NO: 630, SEQ ID NO: 634 or SEQ ID NO: 638.In some aspects, the light chain FWR2 comprises the amino acid sequenceof SEQ ID NO: 234, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO: 281, SEQID NO: 283, SEQ ID NO: 285, SEQ ID NO: 287, SEQ ID NO: 289, SEQ ID NO:291, SEQ ID NO: 293, SEQ ID NO: 295, SEQ ID NO: 297, SEQ ID NO: 438, SEQID NO: 444, SEQ ID NO: 448, SEQ ID NO: 452, SEQ ID NO: 456, SEQ ID NO:460, SEQ ID NO: 464, SEQ ID NO: 492, SEQ ID NO: 498, SEQ ID NO: 502, SEQID NO: 506, SEQ ID NO: 510, SEQ ID NO: 584, SEQ ID NO: 592, SEQ ID NO:593, SEQ ID NO: 609, SEQ ID NO: 615, SEQ ID NO: 619, SEQ ID NO: 623, SEQID NO: 627, SEQ ID NO: 631, SEQ ID NO: 635 or SEQ ID NO: 639 and in someaspects, the light chain FWR2 comprises an amino acid sequence having atleast about 85%, at least about 90%, at least about 92%, at least about93%, at least about 94%, at least about 95%, at least about 96%, atleast about 97%, at least about 98%, or at least about 99% sequenceidentity with the amino acid sequence of SEQ ID NO: 234, SEQ ID NO: 246,SEQ ID NO: 248, SEQ ID NO: 281, SEQ ID NO: 283, SEQ ID NO: 285, SEQ IDNO: 287, SEQ ID NO: 289, SEQ ID NO: 291, SEQ ID NO: 293, SEQ ID NO: 295,SEQ ID NO: 297, SEQ ID NO: 438, SEQ ID NO: 444, SEQ ID NO: 448, SEQ IDNO: 452, SEQ ID NO: 456, SEQ ID NO: 460, SEQ ID NO: 464, SEQ ID NO: 492,SEQ ID NO: 498, SEQ ID NO: 502, SEQ ID NO: 506, SEQ ID NO: 510, SEQ IDNO: 584, SEQ ID NO: 592, SEQ ID NO: 593, SEQ ID NO: 609, SEQ ID NO: 615,SEQ ID NO: 619, SEQ ID NO: 623, SEQ ID NO: 627, SEQ ID NO: 631, SEQ IDNO: 635 or SEQ ID NO: 639. In some aspects, the light chain FWR3comprises the amino acid sequence of SEQ ID NO: 236, SEQ ID NO: 245, SEQID NO: 265, SEQ ID NO: 266, SEQ ID NO: 267, SEQ ID NO: 271, SEQ ID NO:272, SEQ ID NO: 274, SEQ ID NO: 276, SEQ ID NO: 277, SEQ ID NO: 278, SEQID NO: 279, SEQ ID NO: 280, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO:286, SEQ ID NO: 288, SEQ ID NO: 290, SEQ ID NO: 292, SEQ ID NO: 294, SEQID NO: 296, SEQ ID NO: 298, SEQ ID NO: 300, SEQ ID NO: 302, SEQ ID NO:304, SEQ ID NO: 306, SEQ ID NO: 308, SEQ ID NO: 310, SEQ ID NO: 312, SEQID NO: 314, SEQ ID NO: 316, SEQ ID NO: 318, SEQ ID NO: 320, SEQ ID NO:323, SEQ ID NO: 327, SEQ ID NO: 331, SEQ ID NO: 335, SEQ ID NO: 339, SEQID NO: 343, SEQ ID NO: 347, SEQ ID NO: 351, SEQ ID NO: 355, SEQ ID NO:359, SEQ ID NO: 363, SEQ ID NO: 367, SEQ ID NO: 371, SEQ ID NO: 375, SEQID NO: 379, SEQ ID NO: 383, SEQ ID NO: 387, SEQ ID NO: 440, SEQ ID NO:445, SEQ ID NO: 449, SEQ ID NO: 453, SEQ ID NO: 457, SEQ ID NO: 461, SEQID NO: 465, SEQ ID NO: 494, SEQ ID NO: 499, SEQ ID NO: 503, SEQ ID NO:507, SEQ ID NO: 511, SEQ ID NO: 586, SEQ ID NO: 611, SEQ ID NO: 616, SEQID NO: 620, SEQ ID NO: 624, SEQ ID NO: 628, SEQ ID NO: 632, SEQ ID NO:636 or SEQ ID NO: 640, and in some aspects, the light chain FWR3comprises an amino acid sequence having at least about 85%, at leastabout 90%, at least about 92%, at least about 93%, at least about 94%,at least about 95%, at least about 96%, at least about 97%, at leastabout 98%, or at least about 99% sequence identity with the amino acidsequence of SEQ ID NO: 236, SEQ ID NO: 245, SEQ ID NO: 265, SEQ ID NO:266, SEQ ID NO: 267, SEQ ID NO: 271, SEQ ID NO: 272, SEQ ID NO: 274, SEQID NO: 276, SEQ ID NO: 277, SEQ ID NO: 278, SEQ ID NO: 279, SEQ ID NO:280, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO: 286, SEQ ID NO: 288, SEQID NO: 290, SEQ ID NO: 292, SEQ ID NO: 294, SEQ ID NO: 296, SEQ ID NO:298, SEQ ID NO: 300, SEQ ID NO: 302, SEQ ID NO: 304, SEQ ID NO: 306, SEQID NO: 308, SEQ ID NO: 310, SEQ ID NO: 312, SEQ ID NO: 314, SEQ ID NO:316, SEQ ID NO: 318, SEQ ID NO: 320, SEQ ID NO: 323, SEQ ID NO: 327, SEQID NO: 331, SEQ ID NO: 335, SEQ ID NO: 339, SEQ ID NO: 343, SEQ ID NO:347, SEQ ID NO: 351, SEQ ID NO: 355, SEQ ID NO: 359, SEQ ID NO: 363, SEQID NO: 367, SEQ ID NO: 371, SEQ ID NO: 375, SEQ ID NO: 379, SEQ ID NO:383, SEQ ID NO: 387, SEQ ID NO: 440, SEQ ID NO: 445, SEQ ID NO: 449, SEQID NO: 453, SEQ ID NO: 457, SEQ ID NO: 461, SEQ ID NO: 465, SEQ ID NO:494, SEQ ID NO: 499, SEQ ID NO: 503, SEQ ID NO: 507, SEQ ID NO: 511, SEQID NO: 586, SEQ ID NO: 611, SEQ ID NO: 616, SEQ ID NO: 620, SEQ ID NO:624, SEQ ID NO: 628, SEQ ID NO: 632, SEQ ID NO: 636 or SEQ ID NO: 640.In some aspects, the light chain FWR4 comprises the amino acid sequenceof SEQ ID NO: 238, SEQ ID NO: 442, SEQ ID NO: 446, SEQ ID NO: 450, SEQID NO: 454, SEQ ID NO: 458, SEQ ID NO: 462, SEQ ID NO: 496, SEQ ID NO:500, SEQ ID NO: 504, SEQ ID NO: 508, SEQ ID NO: 512, SEQ ID NO: 588, SEQID NO: 613, SEQ ID NO: 617, SEQ ID NO: 621, SEQ ID NO: 625, SEQ ID NO:629, SEQ ID NO: 633, SEQ ID NO: 637 or SEQ ID NO: 641 and in someaspects, the light chain FWR4 comprises an amino acid sequence having atleast about 85%, at least about 90%, at least about 92%, at least about93%, at least about 94%, at least about 95%, at least about 96%, atleast about 97%, at least about 98%, or at least about 99% sequenceidentity with the amino acid sequence of SEQ ID NO: 238, SEQ ID NO: 442,SEQ ID NO: 446, SEQ ID NO: 450, SEQ ID NO: 454, SEQ ID NO: 458, SEQ IDNO: 462, SEQ ID NO: 496, SEQ ID NO: 500, SEQ ID NO: 504, SEQ ID NO: 508,SEQ ID NO: 512, SEQ ID NO: 588, SEQ ID NO: 613, SEQ ID NO: 617, SEQ IDNO: 621, SEQ ID NO: 625, SEQ ID NO: 629, SEQ ID NO: 633, SEQ ID NO: 637or SEQ ID NO: 641. It will be understood that antibodies comprisingamino acid changes in the light chain framework region(s) (FWR1, FWR2,FWR3, and/or FWR4) retain the capability to specifically bind to CD38.The retained CD38 specific binding activity (including affinity) ispreferably about the same as the binding activity (including affinity)of an antibody without any amino acid changes in any light chainframework region(s), although the binding activity (including affinity)may be lesser or greater than an antibody without any amino acid changesin any light chain framework region(s).

In some aspects, the light chain CDR1 comprises the amino acid sequenceof SEQ ID NO: 233, SEQ ID NO: 250, SEQ ID NO: 525, SEQ ID NO: 255, SEQID NO: 262, SEQ ID NO: 263, SEQ ID NO: 319, SEQ ID NO: 322, SEQ ID NO:325, SEQ ID NO: 329, SEQ ID NO: 333, SEQ ID NO: 337, SEQ ID NO: 341, SEQID NO: 345, SEQ ID NO: 349, SEQ ID NO: 353, SEQ ID NO: 357, SEQ ID NO:361, SEQ ID NO: 365, SEQ ID NO: 369, SEQ ID NO: 373, SEQ ID NO: 377, SEQID NO: 381, SEQ ID NO: 385, SEQ ID NO: 437, SEQ ID NO: 491, SEQ ID NO:583, SEQ ID NO: 589, SEQ ID NO: 590, SEQ ID NO: 608, or SEQ ID NO: 696,and in some aspects, the light chain CDR1 comprises an amino acidsequence having at least about 85%, at least about 90%, at least about92%, at least about 93%, at least about 94%, at least about 95%, atleast about 96%, at least about 97%, at least about 98%, or at leastabout 99% sequence identity with the amino acid sequence of SEQ ID NO:233, SEQ ID NO: 250, SEQ ID NO: 525, SEQ ID NO: 255, SEQ ID NO: 262, SEQID NO: 263, SEQ ID NO: 319, SEQ ID NO: 322, SEQ ID NO: 325, SEQ ID NO:329, SEQ ID NO: 333, SEQ ID NO: 337, SEQ ID NO: 341, SEQ ID NO: 345, SEQID NO: 349, SEQ ID NO: 353, SEQ ID NO: 357, SEQ ID NO: 361, SEQ ID NO:365, SEQ ID NO: 369, SEQ ID NO: 373, SEQ ID NO: 377, SEQ ID NO: 381, SEQID NO: 385, SEQ ID NO: 437, SEQ ID NO: 491, SEQ ID NO: 583, SEQ ID NO:589, SEQ ID NO: 590, SEQ ID NO: 608, or SEQ ID NO: 696. In some aspects,the light chain CDR2 comprises the amino acid sequence of SEQ ID NO:235, SEQ ID NO: 249, SEQ ID NO: 253, SEQ ID NO: 264, SEQ ID NO: 299, SEQID NO: 301, SEQ ID NO: 303, SEQ ID NO: 305, SEQ ID NO: 307, SEQ ID NO:309, SEQ ID NO: 311, SEQ ID NO: 313, SEQ ID NO: 315, SEQ ID NO: 317, SEQID NO: 326, SEQ ID NO: 330, SEQ ID NO: 334, SEQ ID NO: 338, SEQ ID NO:342, SEQ ID NO: 346, SEQ ID NO: 350, SEQ ID NO: 354, SEQ ID NO: 358, SEQID NO: 362, SEQ ID NO: 366, SEQ ID NO: 370, SEQ ID NO: 374, SEQ ID NO:378, SEQ ID NO: 382, SEQ ID NO: 386, SEQ ID NO: 439, SEQ ID NO: 493, SEQID NO: 585, SEQ ID NO: 591, SEQ ID NO: 605, SEQ ID NO: 610 or SEQ ID NO:747, and in some aspects, the light chain CDR2 comprises an amino acidsequence having at least about 85%, at least about 90%, at least about92%, at least about 93%, at least about 94%, at least about 95%, atleast about 96%, at least about 97%, at least about 98%, or at leastabout 99% sequence identity with the amino acid sequence of SEQ ID NO:235, SEQ ID NO: 249, SEQ ID NO: 253, SEQ ID NO: 264, SEQ ID NO: 299, SEQID NO: 301, SEQ ID NO: 303, SEQ ID NO: 305, SEQ ID NO: 307, SEQ ID NO:309, SEQ ID NO: 311, SEQ ID NO: 313, SEQ ID NO: 315, SEQ ID NO: 317, SEQID NO: 326, SEQ ID NO: 330, SEQ ID NO: 334, SEQ ID NO: 338, SEQ ID NO:342, SEQ ID NO: 346, SEQ ID NO: 350, SEQ ID NO: 354, SEQ ID NO: 358, SEQID NO: 362, SEQ ID NO: 366, SEQ ID NO: 370, SEQ ID NO: 374, SEQ ID NO:378, SEQ ID NO: 382, SEQ ID NO: 386, SEQ ID NO: 439, SEQ ID NO: 493, SEQID NO: 585, SEQ ID NO: 591, SEQ ID NO: 605, SEQ ID NO: 610 or SEQ ID NO:747. In some aspects, the light chain CDR3 comprises the amino acidsequence of SEQ ID NO: 237, SEQ ID NO: 244, SEQ ID NO: 251, SEQ ID NO:254, SEQ ID NO: 256, SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 268, SEQID NO: 269, SEQ ID NO: 270, SEQ ID NO: 273, SEQ ID NO: 275, SEQ ID NO:321, SEQ ID NO: 324, SEQ ID NO: 328, SEQ ID NO: 332, SEQ ID NO: 336, SEQID NO: 340, SEQ ID NO: 344, SEQ ID NO: 348, SEQ ID NO: 352, SEQ ID NO:356, SEQ ID NO: 360, SEQ ID NO: 364, SEQ ID NO: 368, SEQ ID NO: 372, SEQID NO: 376, SEQ ID NO: 380, SEQ ID NO: 384, SEQ ID NO: 388, SEQ ID NO:441, SEQ ID NO: 495, SEQ ID NO: 587, SEQ ID NO: 594, SEQ ID NO: 595, SEQID NO: 596, SEQ ID NO: 597, SEQ ID NO: 598, SEQ ID NO: 599, SEQ ID NO:600, SEQ ID NO: 601, SEQ ID NO: 602, SEQ ID NO: 603, SEQ ID NO: 604, SEQID NO: 606 or SEQ ID NO: 612, and in some aspects, the light chain CDR3comprises an amino acid sequence having at least about 85%, at leastabout 90%, at least about 92%, at least about 93%, at least about 94%,at least about 95%, at least about 96%, at least about 97%, at leastabout 98%, or at least about 99% sequence identity with the amino acidsequence of SEQ ID NO: 237, SEQ ID NO: 244, SEQ ID NO: 251, SEQ ID NO:254, SEQ ID NO: 256, SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 268, SEQID NO: 269, SEQ ID NO: 270, SEQ ID NO: 273, SEQ ID NO: 275, SEQ ID NO:321, SEQ ID NO: 324, SEQ ID NO: 328, SEQ ID NO: 332, SEQ ID NO: 336, SEQID NO: 340, SEQ ID NO: 344, SEQ ID NO: 348, SEQ ID NO: 352, SEQ ID NO:356, SEQ ID NO: 360, SEQ ID NO: 364, SEQ ID NO: 368, SEQ ID NO: 372, SEQID NO: 376, SEQ ID NO: 380, SEQ ID NO: 384, SEQ ID NO: 388, SEQ ID NO:441, SEQ ID NO: 495, SEQ ID NO: 587, SEQ ID NO: 594, SEQ ID NO: 595, SEQID NO: 596, SEQ ID NO: 597, SEQ ID NO: 598, SEQ ID NO: 599, SEQ ID NO:600, SEQ ID NO: 601, SEQ ID NO: 602, SEQ ID NO: 603, SEQ ID NO: 604, SEQID NO: 606 or SEQ ID NO: 612. It will be understood that antibodiescomprising amino acid changes in the light chain complementaritydetermining region(s) (CDR1, CDR2, and/or CDR3) retain the capability tospecifically bind to CD38. The retained CD38 specific binding activity(including affinity) is preferably about the same as the bindingactivity (including affinity) of an antibody without any amino acidchanges in any light chain complementarity determining region(s),although the binding activity (including affinity) may be lesser orgreater than an antibody without any amino acid changes in any lightchain complementarity determining region(s).

In some aspects, the antibody comprises particular heavy and light chainpairs. The heavy chains having the amino acid sequences of SEQ ID NO:659 may be paired with any light chains having the amino acid sequencesof SEQ ID NO: 664, or the heavy chains having the amino acid sequencesof SEQ ID NO: 665 may be paired with any light chains having the aminoacid sequences of SEQ ID NO: 666, or the heavy chain having the aminoacid sequences of SEQ ID NO: 736 may be paired with any light chainshaving the amino acid sequences of SEQ ID NO: 664.

Variable heavy and variable light chain pairs may comprise pairs fromthe following table:

Variable Heavy Variable Light SEQ ID NO: SEQ ID NO: Antibody Name (aminoacid) (amino acid) A02.10 19 14 A02.11 20 14 A02.112 34 65 A02.12 34 65A02.13 35 65 A02.16 34 92 A02.17 34 93 A02.18 34 73 A02.19 34 74 A02.213 65 A02.20 34 75 A02.21 34 76 A02.22 34 77 A02.23 34 78 A02.24 34 79A02.25 34 80 A02.26 34 81 A02.27 34 82 A02.28 34 83 A02.29 34 84 A02.317 65 A02.30 34 85 A02.31 34 86 A02.32 34 87 A02.33 34 88 A02.34 34 89A02.35 34 90 A02.36 34 91 A02.37 34 66 A02.38 34 113 A02.39 34 112 A02.418 65 A02.40 111 65 A02.41 110 65 A02.43 110 113 A02.44 111 112 A02.4634 67 A02.47 34 68 A02.48 34 69 A02.49 34 70 A02.5 19 65 A02.50 34 71A02.51 34 72 A02.52 34 94 A02.53 34 95 A02.54 34 96 A02.55 34 97 A02.5634 98 A02.57 34 99 A02.58 34 100 A02.59 34 101 A02.6 20 65 A02.60 34 102A02.61 34 103 A02.62 34 104 A02.63 34 105 A02.64 34 106 A02.65 34 107A02.66 34 108 A02.67 34 109 A02.8 17 14 A02.9 18 14 A10.1 165 161 A10.10174 161 A10.11 175 161 A10.12 176 161 A10.13 177 161 A10.14 178 161A10.15 179 161 A10.16 180 161 A10.17 156 181 A10.18 156 182 A10.19 156183 A10.2 166 161 A10.20 156 184 A10.21 156 185 A10.22 156 186 A10.23156 187 A10.24 156 188 A10.25 156 189 A10.26 156 190 A10.27 156 191A10.28 156 192 A10.29 156 193 A10.3 167 161 A10.30 156 194 A10.31 156195 A10.32 156 196 A10.35 197 161 A10.36 156 198 A10.38 152 161 A10.39152 181 A10.4 168 161 A10.40 152 182 A10.41 152 183 A10.42 152 184A10.43 152 185 A10.44 152 186 A10.45 152 187 A10.46 152 188 A10.47 152189 A10.48 152 190 A10.49 152 191 A10.5 169 161 A10.50 152 192 A10.51152 193 A10.52 152 194 A10.53 152 195 A10.54 152 196 A10.57 152 198A10.59 156 161 A10.6 170 161 A10.7 171 161 A10.8 172 161 A10.9 173 161A10A2.0 (chimeric) 148 157 A10A2.1 149 158 A10A2.10 150 160 A10A2.11 150161 A10A2.12 150 162 A10A2.13 150 163 A10A2.14 150 164 A10A2.15 151 158A10A2.16 151 159 A10A2.17 151 160 A10A2.18 151 161 A10A2.19 151 162A10A2.2 149 159 A10A2.20 151 163 A10A2.21 151 164 A10A2.22 152 158A10A2.23 152 159 A10A2.24 152 160 A10A2.25 152 161 A10A2.26 152 162A10A2.27 152 163 A10A2.28 152 164 A10A2.29 153 158 A10A2.3 149 160A10A2.30 153 159 A10A2.31 153 160 A10A2.32 153 161 A10A2.33 153 162A10A2.34 153 163 A10A2.35 153 164 A10A2.36 154 158 A10A2.37 154 159A10A2.38 154 160 A10A2.39 154 161 A10A2.4 149 161 A10A2.40 154 162A10A2.41 154 163 A10A2.42 154 164 A10A2.43 154 158 A10A2.44 155 159A10A2.45 155 160 A10A2.46 155 161 A10A2.47 155 162 A10A2.48 155 163A10A2.49 155 164 A10A2.5 149 162 A10A2.50 156 158 A10A2.51 156 159A10A2.52 156 160 A10A2.53 156 161 A10A2.54 156 162 A10A2.55 156 163A10A2.56 156 164 A10A2.6 149 163 A10A2.7 149 164 A10A2.8 150 158 A10A2.9150 159 A5D1.0 (chimeric) 114 125 A5D1.1 115 126 A5D1.10 116 129 A5D1.11116 130 A5D1.12 116 131 A5D1.13 117 126 A5D1.14 117 127 A5D1.15 117 128A5D1.16 117 129 A5D1.17 117 130 A5D1.18 117 131 A5D1.19 118 126 A5D1.2115 127 A5D1.20 118 127 A5D1.21 118 128 A5D1.22 118 129 A5D1.23 118 130A5D1.24 118 131 A5D1.25 119 126 A5D1.26 119 127 A5D1.27 119 128 A5D1.28119 129 A5D1.29 119 130 A5D1.3 115 128 A5D1.30 119 131 A5D1.31 120 126A5D1.32 120 127 A5D1.33 120 128 A5D1.34 120 129 A5D1.35 120 130 A5D1.36120 131 A5D1.37 121 126 A5D1.38 121 127 A5D1.39 121 128 A5D1.4 115 129A5D1.40 121 129 A5D1.41 121 130 A5D1.42 121 131 A5D1.43 122 126 A5D1.44122 127 A5D1.45 122 128 A5D1.46 122 129 A5D1.47 122 130 A5D1.48 122 131A5D1.49 123 126 A5D1.5 115 130 A5D1.50 123 127 A5D1.51 123 128 A5D1.52123 129 A5D1.53 123 130 A5D1.54 123 131 A5D1.55 124 126 A5D1.56 124 127A5D1.57 124 128 A5D1.58 124 129 A5D1.59 124 130 A5D1.6 115 131 A5D1.60124 131 A5D1.7 116 126 A5D1.8 116 127 A5D1.9 116 128 A5E8.0 (chimeric)132 143 A5E8.1 133 144 A5E8.10 135 145 A5E8.11 135 146 A5E8.12 135 147A5E8.13 136 144 A5E8.14 136 145 A5E8.15 136 146 A5E8.16 136 147 A5E8.17137 144 A5E8.18 137 145 A5E8.19 137 146 A5E8.2 133 145 A5E8.20 137 147A5E8.21 138 144 A5E8.22 138 145 A5E8.23 138 146 A5E8.24 138 147 A5E8.25139 144 A5E8.26 139 145 A5E8.27 139 146 A5E8.28 139 147 A5E8.29 140 144A5E8.3 133 146 A5E8.30 140 145 A5E8.31 140 146 A5E8.32 140 147 A5E8.33141 144 A5E8.34 141 145 A5E8.35 141 146 A5E8.36 141 147 A5E8.37 142 144A5E8.38 142 145 A5E8.39 142 146 A5E8.4 133 147 A5E8.40 142 147 A5E8.5134 144 A5E8.6 134 145 A5E8.7 134 146 A5E8.8 134 147 A5E8.9 135 144X02.10 19 14 X02.100 13 58 X02.101 13 59 X02.102 13 60 X02.103 13 61X02.104 13 62 X02.105 13 63 X02.106 13 64 X02.107 13 65 X02.108 32 14X02.11 20 14 X02.110 33 14 X02.114 13 660 X02.115 13 661 X02.116 13 662X02.117 13 663 X02.118 34 700 X02.119 34 701 X02.120 728 700 X02.121 729700 X02.122 730 700 X02.123 731 700 X02.124 728 701 X02.125 729 701X02.126 730 701 X02.127 731 701 X02.68 21 14 X02.69 22 14 X02.70 23 14X02.71 24 14 X02.72 25 14 X02.73 26 14 X02.74 27 14 X02.75 28 14 X02.7629 14 X02.77 30 14 X02.78 31 14 X02.8 17 14 X02.80 13 38 X02.81 13 39X02.82 13 40 X02.83 13 41 X02.84 13 42 X02.85 13 43 X02.86 13 44 X02.8713 45 X02.88 13 46 X02.89 13 47 X02.9 18 14 X02.90 13 48 X02.91 13 49X02.92 13 50 X02.93 13 51 X02.94 13 52 X02.95 13 53 X02.96 13 54 X02.9713 55 X02.98 13 56 X02.99 13 57 X10.100 720 706 X10.101 721 706 X10.102722 706 X10.103 723 706 X10.104 739 706 X10.105 740 706 X10.106 741 706X10.107 742 706 X10.108 720 707 X10.109 721 707 X10.110 722 707 X10.111723 707 X10.112 739 707 X10.113 740 707 X10.114 741 707 X10.115 742 707X10.116 720 708 X10.117 721 708 X10.118 722 708 X10.119 723 708 X10.120739 708 X10.121 740 708 X10.122 741 708 X10.123 742 708 X10.124 720 709X10.125 721 709 X10.126 722 709 X10.127 723 709 X10.128 739 709 X10.129740 709 X10.130 741 709 X10.131 742 709 X10.132 720 710 X10.133 721 710X10.134 722 710 X10.135 723 710 X10.136 739 710 X10.137 740 710 X10.138741 710 X10.139 742 710 X10.140 720 711 X10.141 721 711 X10.142 722 711X10.143 723 711 X10.144 739 711 X10.145 740 711 X10.146 741 711 X10.147742 711 X10.60 156 704 X10.61 156 705 X10.62 156 706 X10.63 156 707X10.64 156 708 X10.65 156 709 X10.66 156 710 X10.67 156 711 X10.68 720161 X10.69 721 161 X10.70 722 161 X10.71 723 161 X10.72 739 161 X10.73740 161 X10.74 741 161 X10.75 742 161 X10.76 152 704 X10.77 152 705X10.78 152 706 X10.79 152 707 X10.80 152 708 X10.81 152 709 X10.82 152710 X10.83 152 711 X10.84 720 704 X10.85 721 704 X10.86 722 704 X10.87723 704 X10.88 739 704 X10.89 740 704 X10.90 741 704 X10.91 742 704X10.92 720 705 X10.93 721 705 X10.94 722 705 X10.95 723 705 X10.96 739705 X10.97 740 705 X10.98 741 705 X10.99 742 705 X910/12-HC-L0- 110 112IFN-alpha (A145D) IgG4 X913/15-HC-L0- 111 113 IFN-alpha (A145D) IgG4

The antibodies may be fused to attenuated ligands, for example, to formantibody-attenuated ligand constructs, which show an elevatedantigen-specificity index with respect to activating signaling pathwaysdue to the action of the attenuated ligand on a cell surface receptor.These constructs are based on the observation that, in the context of anantibody-ligand construct, the ligand portion can be mutated in such away that the ligand activity on antigen-negative cells is dramaticallyattenuated, while the ligand activity on antigen-positive cells is onlymodestly, if at all, attenuated. Such constructs display one, two,three, four or five orders of magnitude greater potency onantigen-positive cells compared to antigen negative cells than does thefree ligand. In some aspects, the antibody-attenuated ligand constructretains at least 1%, at least 10%, at least 20%, at least 30%, at least40% or at least 50% of the potency on antigen-positive cells as thenon-attenuated free (i.e., not attached to an antibody) ligand. In someaspects, the antibody-attenuated ligand construct retains at least 30%,at least 50%, at least 75% or at least 90% of the maximal activity ofthe non-attenuated free (i.e. not attached to an antibody) ligand.Maximal activity includes the amount of signaling activity (ordownstream effect thereof) at the high, plateau portion of adose-response curve, where further increases in the agent does notfurther increase the amount of response.

In some aspects, the antibody fusion to and inclusion of an attenuatingmutation(s) in the interferon ligand increases the antigen-specificityindex (ASI) by greater than 10-fold, preferably greater than 50-fold,preferably greater than 100-fold, preferably greater than 1000-fold, orpreferably greater than 10,000 fold, relative to an antibody without afusion. The ASI comprises the fold-increased potency in signalingactivity of the antibody-IFN ligand construct relative to the freenon-mutated polypeptide ligand on target antigen-positive cells,multiplied by the fold decreased potency in signaling activity relativeto the free non-mutated polypeptide ligand on target antigen-negativecells. Potency may be quantitatively represented by the EC₅₀ value,which is the mathematical midpoint of a dose-response curve, in whichthe dose refers to the concentration of ligand or antibody-ligandconstruct in an assay, and response refers to the quantitative responseof the cells to the signaling activity of the ligand at a particulardose. Thus, for example, when a first compound is shown to possess anEC₅₀ (expressed for example in Molar units) that is 10-fold lower than asecond compound's EC₅₀ on the same cells, typically when measured by thesame method, the first compound is said to have a 10-fold higherpotency. Conversely, when a first compound is shown to possess an EC₅₀that is 10-fold higher than a second compound's EC₅₀ on the same cells,typically when measured by the same method, the first compound is saidto have a 10-fold lower potency.

The antibodies are preferably capable of binding to CD38-positive cells.The antibody may bind to a CD38-positive cell with an EC₅₀ value of lessthan about 100 nM. The antibody may bind to a CD38-positive cell with anEC₅₀ value of less than about 75 nM. The antibody may bind to aCD38-positive cell with an EC₅₀ value of less than about 50 nM. Theantibody may bind to a CD38-positive cell with an EC₅₀ value of lessthan about 30 nM. The antibody may bind to a CD38-positive cell with anEC₅₀ value of less than about 25 nM. The antibody may bind to aCD38-positive cell with an EC₅₀ value of less than about 20 nM. Theantibody may bind to a CD38-positive cell with an EC₅₀ value of lessthan about 18 nM. The antibody may bind to a CD38-positive cell with anEC₅₀ value of less than about 15 nM. The antibody may bind to aCD38-positive cell with an EC₅₀ value of less than about 13 nM. Theantibody may bind to a CD38-positive cell with an EC₅₀ value of lessthan about 10 nM.

The interferon joined to the antibody preferably comprises alterationsin its amino acid sequence, including point mutations and/or deletionsthat render the interferon less active in stimulating its respectivereceptors on cells that lack cell surface expression of the CD38 antigento which the antibody binds. A highly preferred variant of interferonalpha comprises an amino acid change at position 168 of the interferonalpha 2b molecule of SEQ ID NO: 7. For example, the amino acid atposition 168, which is an alanine in the parent IFN-alpha2b molecule, ispreferably changed to a glycine (Gly/G) (SEQ ID NO: 650) or asparticacid (Asp/D) (SEQ ID NO: 647). In some aspects, the IFN-alpha2b istruncated at its N-terminus when the IFN-alpha2b is fused to an IgGheavy chain constant domain such as the human IgG1 or human IgG4 heavychain constant domain. The truncated IFN-alpha2b does not have thetwenty three N-terminal amino acids of SEQ ID NO:7 (Met 1 through Gly 23are deleted), and the truncated IFN-alpha2b comprises the amino acidsequence of SEQ ID NO: 648. The truncated IFN-alpha2b may also comprisethe amino acid change at what was formerly position 168, but whichbecomes position 145 in the truncated protein (e.g., alanine 168 becomesalanine 145). In the truncated IFN-alpha2b, the alanine is preferablychanged to a glycine (Gly/G) (SEQ ID NO: 651) or aspartic acid (Asp/D)(SEQ ID NO: 649). Interferon with A145D alteration (SEQ ID NO: 647 orSEQ ID NO: 649) is particularly preferred as the attenuated ligand fusedto the antibodies of the disclosure. Any of these point-mutated,attenuated versions of IFN-alpha may be joined to any antibody describedherein, for example, as an antibody-attenuated interferon construct.

The linkage between the antibody and the interferon preferably comprisesa fusion, for example, a peptide bond between the N- or the C-terminusof the interferon and the N- or C-terminus of the heavy or the lightchain of the antibody. In highly preferred aspects, no linker is presentbetween the antibody and the interferon, and the antibody and interferonare thus directly fused. It is believed that direct fusion, without anintervening linker peptide, provides at least a measurable degree ofattenuation of the interferon protein, and it is also believed that thisattenuation is additive with the attenuation of the interferon proteinthat stems from the mutations introduced into the interferon protein,including those described or exemplified herein.

Polynucleotide sequences that encode antibodies and their subdomains(e.g., FWRs and CDRs) are featured in the disclosure. Polynucleotidesinclude, but are not limited to, RNA, DNA, cDNA, hybrids of RNA and DNA,and single, double, or triple stranded strands of RNA, DNA, or hybridsthereof.

In some aspects, the polynucleotides encode the heavy chain of anantibody that specifically binds to an epitope on CD38. Thepolynucleotide may encode a heavy chain comprising the amino acidsequence of any of SEQ ID NO: 667, SEQ ID NO: 668, SEQ ID NO: 679, SEQID NO: 680, SEQ ID NO: 681, SEQ ID NO: 682, SEQ ID NO: 683, SEQ ID NO:684, SEQ ID NO:, SEQ ID NO: 685, SEQ ID NO: 686, SEQ ID NO: 695, SEQ IDNO: 724, SEQ ID NO: 725, SEQ ID NO: 726, SEQ ID NO: 727, SEQ ID NO: 732,SEQ ID NO: 733, SEQ ID NO: 734, SEQ ID NO: 735, SEQ ID NO: 743, SEQ IDNO: 744, SEQ ID NO: 745 or SEQ ID NO: 746. The polynucleotide may encodea light chain comprising the amino acid sequence of any of SEQ ID NO:669, SEQ ID NO: 670, SEQ ID NO: 671, SEQ ID NO: 672, SEQ ID NO: 673, SEQID NO: 674, SEQ ID NO: 675, SEQ ID NO: 676, SEQ ID NO: 677, SEQ ID NO:678, SEQ ID NO: 688, SEQ ID NO: 689, SEQ ID NO: 690, SEQ ID NO: 691, SEQID NO: 692, SEQ ID NO: 693, SEQ ID NO: 702, SEQ ID NO: 703, SEQ ID NO:712, SEQ ID NO: 713, SEQ ID NO: 714, SEQ ID NO: 715, SEQ ID NO: 716, SEQID NO: 717, SEQ ID NO: 718 or SEQ ID NO: 719. The polynucleotide maycomprise the nucleic acid sequence of any of SEQ ID NO: 667, SEQ ID NO:668, SEQ ID NO: 679, SEQ ID NO: 680, SEQ ID NO: 681, SEQ ID NO: 682, SEQID NO: 683, SEQ ID NO: 684, SEQ ID NO:, SEQ ID NO: 685, SEQ ID NO: 686,SEQ ID NO:695, SEQ ID NO: 724, SEQ ID NO: 725, SEQ ID NO: 726, SEQ IDNO: 727, SEQ ID NO: 732, SEQ ID NO: 733, SEQ ID NO: 734, SEQ ID NO: 735,SEQ ID NO: 743, SEQ ID NO: 744, SEQ ID NO: 745, SEQ ID NO: 746, SEQ IDNO: 669, SEQ ID NO: 670, SEQ ID NO: 671, SEQ ID NO: 672, SEQ ID NO: 673,SEQ ID NO: 674, SEQ ID NO: 675, SEQ ID NO: 676, SEQ ID NO: 677, SEQ IDNO: 678, SEQ ID NO: 688, SEQ ID NO: 689, SEQ ID NO: 690, SEQ ID NO: 691,SEQ ID NO: 692, SEQ ID NO: 693, SEQ ID NO: 702, SEQ ID NO: 703, SEQ IDNO: 712, SEQ ID NO: 713, SEQ ID NO: 714, SEQ ID NO: 715, SEQ ID NO: 716,SEQ ID NO: 717, SEQ ID NO: 718 or SEQ ID NO: 719. The polynucleotide maycomprise a nucleic acid sequence having at least about 80%, at leastabout 85%, at least about 86%, at least about 87%, at least about 88%,at least about 89%, at least about 90%, at least about 91%, at leastabout 92%, at least about 93%, at least about 94%, at least about 95%,at least about 96%, at least about 97%, at least about 98%, or at leastabout 99% sequence identity with any of SEQ ID NO: 667, SEQ ID NO: 668,SEQ ID NO: 679, SEQ ID NO: 680, SEQ ID NO: 681, SEQ ID NO: 682, SEQ IDNO: 683, SEQ ID NO: 684, SEQ ID NO:, SEQ ID NO: 685, SEQ ID NO: 686, SEQID NO:695, SEQ ID NO: 724, SEQ ID NO: 725, SEQ ID NO: 726, SEQ ID NO:727, SEQ ID NO: 732, SEQ ID NO: 733, SEQ ID NO: 734, SEQ ID NO: 735, SEQID NO: 743, SEQ ID NO: 744, SEQ ID NO: 745, SEQ ID NO: 746, SEQ ID NO:669, SEQ ID NO: 670, SEQ ID NO: 671, SEQ ID NO: 672, SEQ ID NO: 673, SEQID NO: 674, SEQ ID NO: 675, SEQ ID NO: 676, SEQ ID NO: 677, SEQ ID NO:678, SEQ ID NO: 688, SEQ ID NO: 689, SEQ ID NO: 690, SEQ ID NO: 691, SEQID NO: 692, SEQ ID NO: 693, SEQ ID NO: 702, SEQ ID NO: 703, SEQ ID NO:712, SEQ ID NO: 713, SEQ ID NO: 714, SEQ ID NO: 715, SEQ ID NO: 716, SEQID NO: 717, SEQ ID NO: 718 or SEQ ID NO: 719, and in some aspects suchvariants preferably encode the same amino acids encoded by thepolynucleotide sequence of SEQ ID NO: 667, SEQ ID NO: 668, SEQ ID NO:679, SEQ ID NO: 680, SEQ ID NO: 681, SEQ ID NO: 682, SEQ ID NO: 683, SEQID NO: 684, SEQ ID NO:, SEQ ID NO: 685, SEQ ID NO: 686, SEQ ID NO:695,SEQ ID NO: 724, SEQ ID NO: 725, SEQ ID NO: 726, SEQ ID NO: 727, SEQ IDNO: 732, SEQ ID NO: 733, SEQ ID NO: 734, SEQ ID NO: 735, SEQ ID NO: 743,SEQ ID NO: 744, SEQ ID NO: 745, SEQ ID NO: 746, SEQ ID NO: 669, SEQ IDNO: 670, SEQ ID NO: 671, SEQ ID NO: 672, SEQ ID NO: 673, SEQ ID NO: 674,SEQ ID NO: 675, SEQ ID NO: 676, SEQ ID NO: 677, SEQ ID NO: 678, SEQ IDNO: 688, SEQ ID NO: 689, SEQ ID NO: 690, SEQ ID NO: 691, SEQ ID NO: 692,SEQ ID NO: 693, SEQ ID NO: 702, SEQ ID NO: 703, SEQ ID NO: 712, SEQ IDNO: 713, SEQ ID NO: 714, SEQ ID NO: 715, SEQ ID NO: 716, SEQ ID NO: 717,SEQ ID NO: 718 or SEQ ID NO: 719. Preferably, the antibodies encoded bythe polynucleotide variants will specifically bind to CD38 with anaffinity about equal to the affinity of the antibody encoded by theparent (non-variant) polynucleotide sequence. Affinity may be measured,for example, according to any technique described or exemplified herein,including techniques described in the Examples. Complements of thepolynucleotide sequences and the variant polynucleotide sequences arealso within the scope of the disclosure.

Also encompassed within the disclosure are vectors comprising thepolynucleotides of the disclosure. The vectors may be expressionvectors. Recombinant expression vectors containing a sequence encoding apolypeptide of interest are thus provided. The expression vector maycontain one or more additional sequences, such as but not limited toregulatory sequences, a selection marker, a purification tag, or apolyadenylation signal. Such regulatory elements may include atranscriptional promoter, enhancers, mRNA ribosomal binding sites, orsequences that control the termination of transcription and translation.

Expression vectors, especially mammalian expression vectors, may includeone or more nontranscribed elements, such as an origin of replication, asuitable promoter and enhancer linked to the gene to be expressed, other5′ or 3′ flanking nontranscribed sequences, 5′ or 3′ nontranslatedsequences (such as necessary ribosome binding sites), a polyadenylationsite, splice donor and acceptor sites, or transcriptional terminationsequences. An origin of replication that confers the ability toreplicate in a specific host may also be incorporated.

The vectors may be used to transform any of a wide array of host cellswell known to those of skill in the art, and preferably host cellscapable of expressing antibodies. Vectors include without limitation,plasmids, phagemids, cosmids, baculoviruses, bacmids, bacterialartificial chromosomes (BACs), yeast artificial chromosomes (YACs), andbaculovirus, as well as other bacterial, eukaryotic, yeast, and viralvectors. Suitable host cells include without limitation CHO cells,HEK293 cells, or any eukaryotic stable cell line known or produced, andalso include bacteria, yeast, and insect cells.

The antibodies may also be produced by hybridoma cells; methods toproduce hybridomas being well known and established in the art.

It has been observed in accordance with the disclosure that wheninterferon alpha ligand, having one or more mutations that substantiallydecrease the affinity of the ligand for an interferon receptor, islinked to an anti-CD38 antibody that targets the mutated interferonalpha ligand to target cells which display the antibody's correspondingantigen, the ligand's activity on target antigen-positive cells ismaintained while the ligand's activity on non-target antigen-negativecells is substantially reduced. The net result is a ligand signalingmolecule that has a much greater potency in activation of its receptorson antigen-positive target cells compared to antigen-negative non-targetcells, which provides a means for reducing toxicity arising fromoff-target ligand activity.

In some aspects, a polypeptide construct comprises an IFN-alpha variantlinked to an anti-CD38 antibody or antigen binding portion thereof. Sucha polypeptide will be capable of exerting with high potency the IFN'santi-proliferative activity on CD38-positive tumor cells while exertinga much lower potency on CD38-negative, non-tumor cells within the body.

The disclosure also provides compositions comprising the antibodies andantibody-attenuated interferon constructs of the disclosure. Thesecompositions can further comprise at least one of any suitableauxiliary, such as, but not limited to one or more, diluents, binders,stabilizers, buffers, salts, lipophilic solvents, preservatives,adjuvants, or other suitable carrier and/or excipient. Pharmaceuticallyacceptable auxiliaries are preferred. The compositions may comprise anyof the antibodies and antibody-attenuated interferon constructsdescribed and/or exemplified herein and an acceptable carrier such as apharmaceutically acceptable carrier. Suitable carriers include any mediathat does not interfere with the biological activity of the antibodyand/or the interferon and preferably is not toxic to a host to which itis administered. The carrier may be an aqueous solution, such as water,saline, or alcohol, or a physiologically compatible buffer, such asHanks's solution, Ringer's solution, or physiological saline buffer. Thecarrier may contain formulatory agents, such as suspending, stabilizingand/or dispersing agents

Pharmaceutical excipients and additives useful in the compositioninclude but are not limited to proteins, peptides, amino acids, lipids,and carbohydrates (e.g., sugars, including monosaccharides, di-, tri-,tetra-, and oligosaccharides; derivatized sugars such as alditols,aldonic acids, esterified sugars and other known sugars; andpolysaccharides or sugar polymers), which can be present singly or incombination, comprising alone or in combination any suitable weight orvolume. Exemplary protein excipients include serum albumin, such ashuman serum albumin (HSA), recombinant human albumin (rHA), gelatin,casein, and other known proteins. Representative amino acids which canalso function in a buffering capacity include alanine, glycine,arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine,lysine, leucine, isoleucine, valine, methionine, phenylalanine, andaspartame. One preferred amino acid is histidine. A second preferredamino acid is arginine.

Carbohydrate excipients suitable for use in the composition include, forexample, monosaccharides, such as fructose, maltose, galactose, glucose,D-mannose, and sorbose; disaccharides, such as lactose, sucrose,trehalose, and cellobiose; polysaccharides, such as raffinose,melezitose, maltodextrins, dextrans, and starches; and alditols, such asmannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol), andmyoinositol. Preferred carbohydrate excipients for use in the disclosureare mannitol, trehalose, and raffinose.

Antibody compositions can also include a buffer or a pH adjusting agent;typically, the buffer is a salt prepared from an organic acid or base.Representative buffers include organic acid salts, such as salts ofcitric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid,succinic acid, acetic acid, or phthalic acid; Tris, tromethaminehydrochloride, or phosphate buffers. Preferred buffers for use in thepresent compositions are organic acid salts, such as citrate.

Additionally, the compositions of the disclosure can include polymericexcipients/additives, such as polyvinylpyrrolidones, ficolls (apolymeric sugar), dextrates (e.g., cyclodextrins, such as2-hydroxypropyl-β-cyclodextrin), polyethylene glycols, antimicrobialagents, antioxidants, antistatic agents, surfactants (e.g., polysorbatessuch as “TWEEN® 20” and “TWEEN® 80”), lipids (e.g., phospholipids, fattyacids), steroids (e.g., cholesterol), and chelating agents (e.g., EDTA).

The compositions may also be formulated in sustained release vehicles ordepot preparations. For example, the compositions may be formulated withsuitable polymeric or hydrophobic materials (for example, as an emulsionin an acceptable oil) or ion exchange resins, or as sparingly solublederivatives, for example, as a sparingly soluble salt. Liposomes andemulsions are well-known examples of delivery vehicles suitable for useas carriers for hydrophobic drugs.

The compositions may be formulated for administration to a subject inany suitable dosage form. The compositions may be formulated for oral,buccal, nasal, transdermal, parenteral, injectable, intravenous,subcutaneous, intramuscular, rectal, or vaginal administrations. Thecompositions may be formulated in a suitable controlled-release vehicle,with an adjuvant, or as a depot formulation.

Preparations for parenteral administration include sterile solutionsready for injection, sterile dry soluble products ready to be combinedwith a solvent just prior to use, including hypodermic tablets, sterilesuspensions ready for injection, sterile dry insoluble products ready tobe combined with a vehicle just prior to use and sterile emulsions.

An anti-CD38-attenuated interferon alpha-2b fusion construct may beused, for example, to inhibit, reduce, decrease, block, or preventproliferation of a cell that expressed CD38 on its surface. In someaspects, methods for inhibiting or reducing proliferation of a cell thatexpresses CD38 on its surface generally comprise contacting a cellexpressing CD38 with an anti-CD38-attenuated interferon alpha-2b fusionconstruct in an amount effective to inhibit or reduce proliferation ofthe cell. The antibody that specifically binds to CD38 may be anyantibody described or exemplified herein. The attenuated interferonalpha 2b may comprise IFN-alpha2b A145D or IFN-alpha2b A145G. The cellmay be a lymphocyte, an autoimmune lymphocyte, or a tumor cell such as aleukemia cell, a multiple myeloma cell, or a lymphoma cell. Theanti-CD38-attenuated interferon alpha-2b fusion construct may becomprised in a composition, for example, with a pharmaceuticallyacceptable carrier and optionally one or more auxiliaries or excipients,including any such carrier, auxiliary, or excipient described orexemplified herein. The methods may be carried out in vitro, ex vivo, invivo, or in situ.

An anti-CD38-attenuated interferon alpha-2b fusion construct may also beused, for example, to induce, facilitate, or enhance apoptosis of a cellthat expressed CD38 on its surface. In some aspects, methods forinducing apoptosis in a cell that expresses CD38 on its surfacegenerally comprise contacting a cell expressing CD38 with ananti-CD38-attenuated interferon alpha-2b fusion construct in an amounteffective to induce apoptosis in the cell. The antibody thatspecifically binds to CD38 may be any antibody described or exemplifiedherein. The attenuated interferon alpha 2b may comprise IFN-alpha2bA145D or IFN-alpha2b A145G. The cell may be a lymphocyte, an autoimmunelymphocyte, or a tumor cell such as a leukemia cell, a multiple myelomacell, or a lymphoma cell. The anti-CD38-attenuated interferon alpha-2bfusion construct may be comprised in a composition, for example, with apharmaceutically acceptable carrier and optionally one or moreauxiliaries or excipients, including any such carrier, auxiliary, orexcipient described or exemplified herein. The methods may be carriedout in vitro, ex vivo, in vivo, or in situ.

An anti-CD38-attenuated interferon alpha-2b fusion construct may also beused to treat a subject having a tumor that comprises and/or ismediated, at least in part, by cells that express CD38 on their surface.In some aspects, methods for treating a tumor comprising cellsexpressing CD38 on their surface generally comprise administering to asubject in need thereof an anti-CD38-attenuated interferon alpha-2bfusion construct in an amount effective to treat the tumor in thesubject. Effective treatment may include, for example, inhibiting orreducing proliferation of CD38-positive cells in the tumor and/orinducing apoptosis of CD38-positive cells in the tumor. The antibodythat specifically binds to CD38 may be any antibody described orexemplified herein. The attenuated interferon alpha 2b may compriseIFN-alpha2b A145D or IFN-alpha2b A145G. The anti-CD38-attenuatedinterferon alpha-2b fusion construct may be comprised in a composition,for example, with a pharmaceutically acceptable carrier and optionallyone or more auxiliaries or excipients, including any such carrier,auxiliary, or excipient described or exemplified herein.

The anti-CD38-attenuated interferon alpha-2b fusion constructs orcomposition comprising such constructs may be administered to the tumorby administering the constructs of composition to the blood. Theanti-CD38-attenuated interferon alpha-2b fusion constructs orcomposition comprising such constructs may be administered such that theconstruct diffuses via blood flow to and/or into the tumor cells. Theconstruct may be internalized by a tumor cell.

Use of an anti-CD38 antibody or anti-CD38 antibody-attenuated interferonalpha-2b fusion construct in the treatment of tumors are provided.Methods for treating tumors with an anti-CD38 antibody or anti-CD38antibody-attenuated interferon alpha-2b fusion construct are provided.Any anti-CD38 antibody or anti-CD38 antibody-attenuated interferonalpha-2b fusion construct described or exemplified herein may be used.Tumors that may be treated include, but are not limited to AIDS relatedcancers, acoustic neuroma, acute lymphocytic leukemia, acute myeloidleukemia, adenocystic carcinoma, adrenocortical cancer, agnogenicmyeloid metaplasia, alopecia, alveolar soft-part sarcoma, anal cancer,angiosarcoma, aplastic anemia, astrocytoma, ataxia-telangiectasia, basalcell carcinoma (skin), bladder cancer, bone cancers, bowel cancer, brainstem glioma, brain and CNS tumors, breast cancer, CNS tumors, carcinoidtumors, cervical cancer, childhood brain tumors, childhood cancer,childhood leukemia, childhood soft tissue sarcoma, chondrosarcoma,choriocarcinoma, chronic lymphocytic leukemia, chronic myeloid leukemia,colorectal cancers, cutaneous T-Cell lymphoma,dermatofibrosarcoma-protuberans, desmoplastic-small-round-cell-tumor,ductal carcinoma, endocrine cancers, endometrial cancer, ependymoma,esophageal cancer, Ewing's sarcoma, extra-hepatic bile duct cancer, eyecancer, eye: melanoma, retinoblastoma, fallopian tube cancer, fanconianemia, fibrosarcoma, gall bladder cancer, gastric cancer,gastrointestinal cancers, gastrointestinal-carcinoid-tumor,genitourinary cancers, germ cell tumors,gestational-trophoblastic-disease, glioma, gynecological cancers,hematological malignancies, hairy cell leukemia, head and neck cancer,hepatocellular cancer, hereditary breast cancer, histiocytosis,Hodgkin's disease, human papillomavirus, hydatidiform mole,hypercalcemia, hypopharynx cancer, intraocular melanoma, islet cellcancer, Kaposi's sarcoma, kidney cancer, Langerhan's-cell-histiocytosis,laryngeal cancer, leiomyosarcoma, leukemia, Li-Fraumeni syndrome, lipcancer, liposarcoma, liver cancer, lung cancer, lymphedema, lymphoma,Hodgkin's lymphoma, non-Hodgkin's lymphoma, male breast cancer,malignant-rhabdoid-tumor-of-kidney, medulloblastoma, melanoma, merkelcell cancer, mesothelioma, metastatic cancer, mouth cancer, multipleendocrine neoplasia, mycosis fungoides, myelodysplastic syndromes,multiple myeloma, myeloproliferative disorders, nasal cancer,nasopharyngeal cancer, nephroblastoma, neuroblastoma, neurofibromatosis,nijmegen breakage syndrome, non-melanoma skin cancer,non-small-cell-lung-cancer-(NSCLC), ocular cancers, esophageal cancer,oral cavity cancer, oropharynx cancer, osteosarcoma, ostomy ovariancancer, pancreas cancer, paranasal cancer, parathyroid cancer, parotidgland cancer, penile cancer, peripheral-neuroectodermal-tumors,pituitary cancer, polycythemia vera, prostate cancer,rare-cancers-and-associated-disorders, renal cell carcinoma,retinoblastoma, rhabdomyosarcoma, Rothmund-Thomson syndrome, salivarygland cancer, sarcoma, schwannoma, Sezary syndrome, skin cancer, smallcell lung cancer (SCLC), small intestine cancer, soft tissue sarcoma,spinal cord tumors, squamous-cell-carcinoma-(skin), stomach cancer,synovial sarcoma, testicular cancer, thymus cancer, thyroid cancer,transitional-cell-cancer-(bladder),transitional-cell-cancer-(renal-pelvis-/-ureter), trophoblastic cancer,urethral cancer, urinary system cancer, uroplakins, uterine sarcoma,uterus cancer, vaginal cancer, vulva cancer,Waldenstrom's-macroglobulinemia and Wilms' tumor. In an embodiment thetumor is selected from a group of multiple myeloma or non-hodgkin'slymphoma.

In preferred aspects, the methods are used for treatment of multiplemyeloma, leukemia, or lymphoma in a subject in need thereof. Suchmethods may further comprise treating the subject with a retinoid, suchas all-trans retinoic acid. In some preferred aspects in which the cellsurface associated antigen is CD38, the tumor or cancer may be selectedfrom multiple myeloma, non-Hodgkin's lymphoma, chronic myelogenousleukemia, chronic lymphocytic leukemia or acute myelogenous leukemia.

An anti-CD38-attenuated interferon alpha-2b fusion construct may becombined with other drugs and/or used in addition to other cancertreatment regimens or modalities such as radiation therapy or surgery.When anti-CD38-attenuated interferon alpha-2b fusion constructs are usedin combination with known therapeutic agents the combination may beadministered either in sequence (either continuously or broken up byperiods of no treatment) or concurrently or as a mixture. In the case ofcancer, there are numerous known anticancer agents that may be used inthis context. Treatment in combination is also contemplated to encompassthe treatment with either the anti-CD38-attenuated interferon alpha-2bfusion construct followed by a known treatment, or treatment with aknown agent followed by treatment with the anti-CD38-attenuatedinterferon alpha-2b fusion construct, for example, as maintenancetherapy. For example, in the treatment of cancer it is contemplated thatthe anti-CD38-attenuated interferon alpha-2b fusion construct may beadministered in combination with an alkylating agent (such asmechlorethamine, cyclophosphamide, chlorambucil, ifosfamidecysplatin, orplatinum-containing alkylating-like agents such as cisplatin,carboplatin and oxaliplatin), an antimetabolite (such as a purine orpyrimidine analogue or an antifolate agent, such as azathioprine andmercaptopurine), an anthracycline (such as Daunorubicin, Doxorubicin,Epirubicinldarubicin, Valrubicin, Mitoxantrone, or anthracyclineanalog), a plant alkaloid (such as a vinca alkaloid or a taxane, such asVincristine, Vinblastine, Vinorelbine, Vindesine, paclitaxel orDosetaxel), a topoisomerase inhibitor (such as a type I or type IItopoisomerase inhibitor), a Podophyllotoxin (such as etoposide orteniposide), or a tyrosine kinase inhibitor (such as imatinibmesylate,Nilotinib, or Dasatinib).

In the case of the treatment of multiple myeloma, ananti-CD38-attenuated interferon alpha-2b fusion construct may beadministered in combination with other suitable therapies, such astreatment of the subject with the administration of steroids such asdexamethasone, proteasome inhibitors (such as bortezomib orcarfilzomib), immunomodulatory drugs (such as thalidomide, lenalidomideor pomalidomide), or induction chemotherapy followed by autologoushematopoietic stem cell transplantation, with or without otherchemotherapeutic agents such as Melphalan hydrochloride or thechemotherapeutic agents listed above.

In the case of the treatment of Hodgkin's lymphoma, ananti-CD38-attenuated interferon alpha-2b fusion construct may beadministered in combination with current therapeutic approaches, such asABVD (Adriamycin (doxorubicin), bleomycin, vinblastine, anddacarbazine), or Stanford V (doxorubicin, bleomycin, vinblastine,vincristine, mechlorethamine, etoposide, prednisone), or BEACOPP(doxorubicin, bleomycin, vincristine, cyclophosphamide, procarbazine,etoposide, prednisone).

In the case of non-Hodgkin's lymphoma or other lymphomas, ananti-CD38-attenuated interferon alpha-2b fusion construct may beadministered in combination current therapeutic approaches. Examples ofdrugs approved for non-Hodgkin lymphoma include Abitrexate(Methotrexate), Adriamycin PFS (Doxorubicin Hydrochloride), AdriamycinRDF (Doxorubicin Hydrochloride), Ambochlorin (Chlorambucil), Amboclorin(Chlorambucil), Arranon (Nelarabine), Bendamustine Hydrochloride, Bexxar(Tositumomab and Iodine I 131 Tositumomab), Blenoxane (Bleomycin),Bleomycin, Bortezomib, Chlorambucil, Clafen (Cyclophosphamide),Cyclophosphamide, Cytoxan (Cyclophosphamide), DenileukinDiftitox,DepoCyt (Liposomal Cytarabine), Doxorubicin Hydrochloride, DTIC-Dome(Dacarbazine), Folex (Methotrexate), Folex PFS (Methotrexate), Folotyn(Pralatrexate), IbritumomabTiuxetan, Istodax (Romidepsin), Leukeran(Chlorambucil), Linfolizin (Chlorambucil), Liposomal Cytarabine,Matulane (Procarbazine Hydrochloride), Methotrexate, Methotrexate LPF(Methotrexate), Mexate (Methotrexate), Mexate-AQ (Methotrexate), Mozobil(Plerixafor), Nelarabine, Neosar (Cyclophosphamide), Ontak(DenileukinDiftitox), Plerixafor, Pralatrexate, Rituxan (Rituximab),Rituximab, Romidepsin, Tositumomab and Iodine I 131 Tositumomab, Treanda(Bendamustine Hydrochloride), Velban (Vinblastine Sulfate), Velcade(Bortezomib), and Velsar (Vinblastine Sulfate), Vinblastine Sulfate,Vincasar PFS (Vincristine Sulfate), Vincristine Sulfate, Vorinostat,Zevalin (IbritumomabTiuxetan), Zolinza (Vorinostat). Examples of drugcombinations used in treating non-Hodgkin lymphoma include CHOP(C=Cyclophosphamide, H=Doxorubicin Hydrochloride (Hydroxydaunomycin),O=Vincristine Sulfate (Oncovin), P=Prednisone); COPP(C=Cyclophosphamide, O=Vincristine Sulfate (Oncovin), P=ProcarbazineHydrochloride, P=Prednisone); CVP (C=Cyclophosphamide, V=VincristineSulfate, P=Prednisone); EPOCH (E=Etoposide, P=Prednisone, O=VincristineSulfate (Oncovin), C=Cyclophosphamide, H=Doxorubicin Hydrochloride(Hydroxydaunomycin)); ICE (I=Ifosfamide, C=Carboplatin, E=Etoposide) andR-CHOP (R=Rituximab, C=Cyclophosphamide, H=Doxorubicin Hydrochloride(Hydroxydaunomycin), O=Vincristine Sulfate (Oncovin), P=Prednisone.

An anti-CD38 antibody, or an anti-CD38-attenuated interferon alpha-2bfusion construct may be used to detect CD38-positive cells, includingCD38-positive tumor cells. In some aspects, they may be used in methodsfor detecting a CD38-positive tumor cell in a tissue sample isolatedfrom a subject, which methods may generally comprise contacting ananti-CD38 antibody, or an anti-CD38-attenuated interferon alpha-2bfusion construct, with a tissue sample isolated from a subject anddetecting a complex of the antibody or construct and a CD38-positivecell in the tissue sample. The tissue sample preferably is blood. Thecell may be a CD38-positive B-cell lymphoma cell, multiple myeloma cell,non-Hodgkin's lymphoma cell, chronic myelogenous leukemia cell, chroniclymphocytic leukemia cell, or acute myelogenous leukemia cell. Themethod may further comprise isolating the tissue sample from thesubject.

The disclosure also features kits comprising any of the antibodies andanti-CD38-attenuated interferon alpha-2b fusion constructs described andexemplified herein. The kits may be used to supply antibodies and otheragents for use in diagnostic, basic research, or therapeutic methods,among others.

In some aspects, a kit comprises an anti-CD38-attenuated interferonalpha-2b fusion construct, the construct optionally comprised in acomposition comprising a pharmaceutically acceptable carrier, andinstructions for using the kit in one or more of a method for inhibitingor reducing proliferation of a tumor cell expressing CD38 on itssurface, a method for inducing apoptosis in a tumor cell expressing CD38on its surface, and/or a method for treating a tumor that comprisesand/or is mediated by cells expressing CD38 on their surface. Suchmethods may be any method described or exemplified herein. The kits maycomprise a pharmaceutically acceptable carrier. The kits may compriseone or more pharmaceutically acceptable auxiliaries and/or one or morepharmaceutically acceptable excipients. In the kits, the anti-CD38antibody may be any antibody described or exemplified herein, and theattenuated interferon alpha-2b may comprise any attenuated interferonalpha-2b described or exemplified herein. The constructs may becomprised in sterile solutions ready for injection or intravenousadministration, or may comprise a sterile, lyophilized form ready to becombined with a carrier just prior to use.

In some aspects, a kit comprises an anti-CD38 antibody and instructionsfor using the kit in a method for detecting CD38-positive cells in asample, including a tissue sample isolated from a subject. The anti-CD38antibody may be any antibody described or exemplified herein. Theantibody may optionally be fused to an attenuated interferon alpha-2bprotein.

The following examples are provided to describe the disclosure ingreater detail. They are intended to illustrate, not to limit, thedisclosure.

Example 1 Optimization of X355/02-HC-L0-IFN-alpha (A145D) IgG4

Other anti-CD38-attenuated IFN fusion proteins are described in PCTApplication No. PCT/AU2012/001323. These include the antibody constructdesignated in the PCT application as X355/02-HC-L0-IFN-alpha (A145D)IgG4. In this specification, X355/02-HC-L0-IFN-alpha (A145D) IgG4 hasbeen renamed as A02.1. The heavy chain sequence of the antibodycomprises the amino acid sequence of SEQ ID NO: 11, and the light chainsequence comprises the amino acid sequence of SEQ ID NO: 12. Thevariable light chain of A02.1 (SEQ ID NO: 14) was co-expressed with itsvariable heavy chain A02.1 (SEQ ID NO: 13) formatted on a human IgG4constant region containing the substitution S228P (EU Numbering) (SEQ IDNO: 3). This antibody is referred to herein as X02.1. A02.1 includes afusion to IFN-alpha2b whilst X02.1 does not, despite both antibodiessharing identical heavy chain and light chains sequences.

A BLAST search (Altschul S F (1997) Nucleic Acids Res. 25:3389-3402)against a database of human germline immunoglobulin genes was performedusing the amino acid sequence of the variable heavy chain of X02.1. Theclosest human germline variable heavy chain gene was IGHV4-61*01 (SEQ IDNO: 16). An alignment of the X02.1 VH and IGHV4-61*01 is shown in FIG.2. The X02.1 variable heavy region differs by eight amino acids from itsclosest germline amino acid sequence. In order to reduce theimmunogenicity of the X02.1 heavy chain variable region, germline aminoacid residue substitutions could be produced at residues where itdiffers from the germline sequence and the resulting antibody variantstested for anti-CD38 binding activity.

Several heavy chain variants of the X02.1 parental sequence are detailedin FIG. 2. These heavy chain variable regions were formatted onto theIgG4 S228P constant region, and co-expressed with the A02.1 light chain.Tables 1a and 1b detail the sequences of the variants tested along withtheir ability to bind human CD38 as assessed using flow cytometry andsurface plasmon resonance (SPR). Briefly, antibody chains weretransiently co-expressed in CHO cells and purified via Protein Achromatography as described in the Example 5. Flow binding assays asdescribed in Example 5 were used to assess the variants. The EC₅₀ of thedose response curve obtained for each antibody is also given in Tables1a and 1b.

TABLE 1a Variable Heavy ARP-1 Chain flow Amino Acid Variable Variablebinding Substitution Heavy Light CD38 assay Antibody (Relative to SEQ IDSEQ ID binding (EC₅₀ in Designation X02.1) NO: NO: by SPR μg/mL) X02.8L74S 17 14 2.30 × 10⁻⁸ 18.3 X02.9 H40P 18 14 2.63 × 10⁻⁸ N/T X02.10T(82A)S 19 14 2.07 × 10⁻⁸ N/T X02.11 L74S, I78F 20 14 2.39 × 10⁻⁸ 18.1R81K, T(82A)S X02.108 I78F 32 14 2.63 × 10⁻⁸ N/T X02.110 R81K 33 14 2.07× 10⁻⁸ N/T N/T—Protein was not able to be purified and was not tested.

TABLE 1b Variable Heavy ARP-1 Chain flow Amino Acid Variable Variablebinding Substitution Heavy Light CD38 assay Antibody (Relative to SEQ IDSEQ ID binding (EC₅₀ in Designation X02.1) NO: NO: by SPR μg/mL) X02.69Q2V 22 14 3.68 × 10⁻¹¹ 16.8 X02.71 I29V 24 14 1.29 × 10⁻¹⁰ 3.5 X02.78S32G 31 14 2.04 × 10⁻¹¹ N/T N/T—Protein not purified or tested.

SPR binding of the variants detailed in Table 1a was evaluatedseparately to those of Table 1b. The K_(D) (M) of the parental antibodyX02.1 ranged from 2.7×10⁻⁸ to 3.78×10⁻¹⁰ in the SPR binding experiments.Flow cytometry binding experiments showed antibodies X02.8, X02.11,X02.69 and X02.71 bound strongly to the CD38 positive cell line ARP-1.

Antibodies with the above amino acid substitutions were subsequentlyexplored in the context of a fusion protein through conjugation toattenuated IFN-alpha2b (termed A02 when linked to IFN, with the numberfollowing the decimal representing the same variant having the X02designation). These heavy chain variable regions were formatted onto anIgG4 constant region comprising the substitution S228P fused to A145Dattenuated IFN-alpha2b and co-expressed in CHO or HEK cells with theA02.1 light chain as described in the Example 5. Proteins that weresuccessfully purified from cell supernatant were then tested in a flowbinding assay to the cell line ARP-1. The EC₅₀ value of the doseresponse curve for each antibody is given in Table 2. Allantibody-attenuated IFN fusion constructs tested bound to the CD38positive cell line ARP-1. It was observed that heavy chain variant X02.9(not fused to IFN) could not easily be purified whereas an identicalvariant fused to IFN (A02.9) was purified. In some cases, attenuated IFNfusion proteins could be expressed and purified, when the equivalentmonoclonal antibody appeared more difficult to be expressed and/orpurify.

TABLE 2 Variable Heavy Chain CD38 ARP-1 flow Anti-CD38- Amino AcidVariable Variable Protein binding binding attenuated Substitution HeavyLight Acapture by by SPR assay IFN fusion (Relative to SEQ ID SEQ ID SPR(RU) at (EC₅₀ in protein A02.1) NO: NO: (RU)* 350 sec* μg/mL) A02.8 L74S17 14 4697 833 1.9 A02.9 H40P 18 14 4718 841 1.0 A02.10 T(82A)S 19 144647 804 1.5 A02.11 L74S, I78F, 20 14 4483 827 3.5 R81K, T(82A)S *Note:The amount of Anti-CD38 attenuated IFN fusion protein in the cellculture supernatant is indicated by the Protein A capture by SPR. TheCD38 binding by SPR refers to the amount of CD38 that remains bound tothe surface after 350 seconds of the dissociation phase.

BLAST searches using the amino acid sequence of the A02.1 variable lightchain were performed against the database of human germlineimmunoglobulin genes. The closest human germline variable light chaingene was IGLV5-37*01. An amino acid sequence alignment of A02.1VL andIGLV5-37*01 is given in FIG. 3. This alignment illustrates a 12 aminoacid difference between these sequences.

Several amino acid substitutions were made in the X02.1 variable lightchain. These substitutions are shown in FIG. 3. Co-expression of theselight chain variable regions with the X02.1 variable heavy chainformatted onto an IgG4 constant region containing the substitution S228Pwas performed in CHO cells as described in Example 5.

Antibodies purified from CHO cell supernatants were subsequently testedin flow cytometry-based binding assays to the CD38 positive cell lineARP-1. Table 3 details ECs50 values of the dose response curve obtainedfor each antibody.

TABLE 3 Variable ARP-1 Light Chain flow Amino Acid Variable VariableCD38 binding Substitution Heavy Light binding assay Protein A Antibody(Relative to SEQ ID SEQ ID by SPR (EC₅₀ in HPLC Designation A02.1) NO:NO: (KD) μg/mL) mg/L X02.95 A2P 13 53 4.40 × 10⁻¹² N/T 11.1 X02.96 A8P13 54 2.50 × 10⁻¹⁰ 30.0 19.9 X02.97 L11S 13 55 7.51 × 10⁻¹² Low 15.0Binding X02.98 R29S 13 56 5.84 × 10⁻¹² N/T 8.7 X02.99 Y30S 13 57 3.21 ×10⁻¹²  4.4 11.5 X02.100 H(54A)D 13 58 1.60 × 10⁻⁷  Low 18.9 BindingX02.101 V(66B)A 13 59 2.89 × 10⁻¹¹ 13.7 21.0 X02.102 T68A 13 60 1.20 ×10⁻¹⁰ 20.4 18.0 X02.103 S70T 13 61 1.28 × 10⁻⁹   9.4 27.9 X02.104 T90I13 62  1.0 × 10⁻⁸  9.7 23.1 X02.105 S92P 13 63 3.31 × 10⁻⁸   12.7* 19.3X02.106 G95A 13 64 4.47 × 10⁻⁸  N/T 11.6 N/T - Protein not purified ornot tested. Low Binding - Minimal binding observed, not sufficient foran EC₅₀ value. *Antibody was tested in a flow binding assay against H929cell line. Reported value is the EC₅₀ in μg/mL.

Antibodies X02.96, X02.99, X02.101, X02.102, X02.103 and X02.104 boundstrongly to the CD38 positive ARP-1 cell line. X02.105 was able to bindstrongly to the CD38 positive, H929 cell line.

Amino acid sequence analysis of the variable heavy chain sequence ofX02.1 and A02.1 identified amino acids that could potentially undergooxidation or isomerization. These include a potential isomerization siteat D101 and a potential oxidation site at M(100C). To remove thepotential isomerization and oxidation sites, amino acid substitutionswere made as follows: D(101)E (SEQ ID NO: 30), M(100C)L (SEQ ID NO: 29)and the combination of both D(101)E and M(100C)L (SEQ ID NO: 27) (FIG.2). Antibodies were made with combinations of these amino acidsubstitutions in the variable heavy chain as shown in Table 4. Antibodyheavy chain variable regions were formatted with an IgG4 constant regioncontaining the substitution S228P and co-expressed with the A02.1 lightchain in CHO cells. Antibodies were then purified by Protein Achromatography and screened for binding to ARP-1 cells by flowcytometry. The binding data obtained is shown in Table 4.

TABLE 4 Variable ARP-1 Heavy Chain flow Amino Acid Variable VariableCD38 binding Substitution Heavy Light binding assay Protein A Antibody(Relative to SEQ ID SEQ ID by SPR (EC₅₀ in HPLC Designation X02.1) NO:NO: (KD) μg/mL) mg/L X02.76 M(100C)L 29 14 1.58 × 10⁻¹³ 4.1 24.8 X02.77D101E 30 14 9.11 × 10⁻¹² 3.7 15.6 X02.74 M(100C)L, D101E 27 14 6.85 ×10⁻¹¹ N/T 21.8 N/T - Protein was not purified or not tested.

Antibodies X02.76 and X02.77 maintained their strong binding to theARP-1 cell line indicating that the amino acids substitutions to removethe potential oxidation and isomerization sites in the X02.1 and A02.1heavy chain had little impact on their CD38 binding activity. Combiningthese substitutions to form antibody X02.74 resulted in an antibody thatdid not purify using the protocol in Example 5.

Amino acid analysis of the variable light chain sequence of X02.1 andA02.1 identified amino acids that could potentially undergo oxidation ordeamidation. These included a potential deamidation site at N69 andpotential oxidation site at M89. Additionally a putative N-linkedglycosylation site was predicted to exist within CDR3 of the light chainat position N94. The presence of N-linked glycans can causeheterogeneity in therapeutic proteins, complicating development. Toremove these potential issues the following point variants weresynthesized: N69A (SEQ ID NO: 39), M89L (SEQ ID NO: 52) and M891 (SEQ IDNO: 51), N94T (SEQ ID NO: 48), N94Q (SEQ ID NO: 38), G95P (SEQ ID NO:50) and S96A (SEQ ID NO: 45) (see FIG. 3). Antibodies were generated byco-expression of the heavy- and light chains in CHO cells as detailed inTable 5. Antibodies were purified by Protein A chromatography andscreened for binding to ARP-1 cells by flow cytometry. The binding dataobtained is presented in Table 5.

TABLE 5 Variable ARP-1 Light Chain flow Amino Acid Variable Variablebinding Substitution Heavy Light CD38 assay Protein A Antibody (Relativeto SEQ ID SEQ ID binding (EC₅₀ in HPLC Designation A02.1) NO: NO: by SPRμg/mL) mg/L X02.81 N69A 13 39 2.67 × 10⁻¹⁰ N/T 12.6 X02.93 M89I 13 512.56 × 10⁻¹¹ 18.7 20.0 X02.94 M89L 13 52 3.48 × 10⁻¹² 8.7 18.9 X02.90N94T 13 48 5.52 × 10⁻¹⁰ 26.2 23.0 X02.80 N94Q 13 38 1.44 × 10⁻⁹  13.230.3 X02.92 G95P 13 50 Low Binding Low 18.1 Binding X02.87 S96A 13 451.99 × 10⁻⁹  37.5 18.9 N/T - Protein was not purified or tested LowBinding - Minimal binding observed, not sufficient for an EC₅₀ or KDvalue.

X02.94 bound the CD38 positive cell line ARP-1 indicating that thesubstitution M89L had little impact on CD38 binding activity. Thesubstitution N94Q in antibody X02.80 removed the potential N-linkedglycosylation motif with minimal impact on CD38 binding activity asmeasure by flow cytometry (Table 5). Other substitutions that removethis glycosylation motif either resulted in antibodies that could noteasily be purified or antibodies that exhibited attenuated binding tothe CD38 positive cell line ARP-1. The potential deamidation site atposition 69 was removed through substitution to alanine, though thisantibody (X02.81) was not easily purified.

Other antibodies tested that comprised X02.1 variable heavy chainvariants are listed in Table 6. These heavy chain variable regions wereformatted on an IgG4 constant region containing an S228P substitution.These heavy chains were co-expressed with the A02.1 light chain in CHOcells. The antibodies were expressed and the resulting antibodies testedin flow cytometry-based assays for binding to the CD38-positive cellARP-1. All variable heavy chain substitutions with the exception of T23K(SEQ ID NO:21; X02.68) had minimal impact on binding to the CD38positive cell line ARP-1 in flow cytometry-based assays.

TABLE 6 Variable ARP-1 Heavy Chain flow Amino Acid Variable Variablebinding Substitution Heavy Light CD38 assay Protein A Antibody (Relativeto SEQ ID SEQ ID binding (EC₅₀ in HPLC Designation X02.1) NO: NO: by SPRμg/mL) mg/L X02.73 S19K 26 14 3.85 × 10⁻¹⁰ 4.3 20.6 X02.68 T23K 21 141.06 × 10⁻¹¹ N/T 17.0 X02.70 V71R 23 14 3.54 × 10⁻⁹  7.2 32.8 X02.75T73K 28 14 6.38 × 10⁻¹⁰ 4.9 17.8 X02.72 T83R 25 14 1.63 × 10⁻⁹  3.6 30.6N/T - Protein not purified or tested.

Antibodies comprising other light chain variable region substitutions inthe X02.1 sequence were also produced. These variant light chains werecombined with the X02.1 heavy chain formatted onto an IgG4 constantregion containing the substitution S228P and expressed in CHO cells asdescribed in Example 5. A summary of the heavy- and light chains used toproduce these antibody variants is given in Table 7. Antibodies X02.83,X02.85, X02.91, X02.82 bound strongly to the CD38 positive cell lineARP-1.

TABLE 7 Variable Light Chain ARP-1 flow Amino Acid Variable Variablebinding Substitution Heavy Light CD38 assay Protein A Antibody (Relativeto SEQ ID SEQ ID binding (EC₅₀ in HPLC Designation A02.1) NO: NO: viaSPR μg/mL) mg/L X02.83 E17A 13 41 2.69 × 10⁻⁹  8.2 32.8 X02.86 D(27A)G13 44 4.28 × 10⁻⁹  120.4  30.0 X02.85 ΔD(L66A)*** 13 43 2.70 × 10⁻¹⁰ 6.317.6 ΔV(L66B)*** X02.107 E83I, D85T 13 65  2.47 × 10^(−8##) N/T 10.0*X02.91 P26R 13 49 7.07 × 10⁻¹⁰ 17.6  21.1 X02.88 N32R 13 46 Low bindingLow Binding 33.5 X02.82 Y49R 13 40 N/T 3.8 27.9 X02.89 Y51R 13 47 Lowbinding No Binding 25.7 X02.84 Y49R, Y51R 13 42 Low binding No Binding35.0 N/T - Protein was not purified or tested. Low Binding - Minimalbinding observed, not sufficient for an EC50 value ***Δ indicates thatthis amino acid present in A02.1 light chain was removed from thissequence *estimated protein value based on protein A capture level bySPR ^(##)The SPR binding for X02.107 was evaluated in a separateexperiment in which the KD of the parental antibody X02.1 was 2.7 ×10⁻⁸. The KD of the parental antibody X02.1 is 3.78 × 10⁻¹⁰ in the SPRbinding experiment for all other antibodies tested.

Substitutions causing little impact on CD38 binding activity and thepurification of X02 variant antibodies were subsequently produced asarmed antibodies through fusion to A145D attenuated IFN-alpha2b. X02.1light chain substitutions were combined and the resulting variantsco-expressed with point- and combinatorial variants of the X02.1 heavychain in HEK293E cells, as listed in Table 8. These antibodies wereprimarily focused on removing the potential X02.1 light chaindeamidation site, an oxidation site from CDR3 of the X02.1 heavy chainand a putative strong MHC Class II binding peptide from framework region3 of the X02.1 heavy chain predicted via in silico analyses (Epibase,Lonza, UK), FIG. 4.

TABLE 8 Variable Heavy Variable Light Anti-CD38- Chain Amino Chain Aminoattenuated Acid Variable Acid Variable IFN Substitution HeavySubstitution Light fusion (Relative to SEQ (Relative to SEQ proteinA02.1) ID NO: A02.1) ID NO: A02.2 None 13 E83I, D85T 65 A02.3 L74S 17E83I, D85T 65 A02.4 H40P 18 E83I, D85T 65 A02.5 T(82A)S 19 E83I, D85T 65A02.6 L74S, I78F, 20 E83I, D85T 65 R81K, T(82A)S A02.12 L74S, I78F, 34E83I, D85T 65 R81K, T(82A)S, M(100C)L A02.13 H40P, L74S, 35 E83I, D85T65 I78F, R81K, T(82A)S A02.37 L74S, I78F, 34 E83I, D85T, 66 R81K,T(82A)S, M89L M(100C)L A02.46 L74S, I78F, 34 E83I, D85T, 67 R81K,T(82A)S, N69Q M(100C)L A02.47 L74S, I78F, 34 E83I, D85T, 68 R81K,T(82A)S, N69T M(100C)L A02.48 L74S, I78F, 34 E83I, D85T, 69 R81K,T(82A)S, N69G M(100C)L A02.49 L74S, I78F, 34 E83I, D85T, 70 R81K,T(82A)S, N69H M(100C)L A02.50 L74S, I78F, 34 E83I, D85T, 71 R81K,T(82A)S, N69K M(100C)L A02.51 L74S, I78F, 34 E83I, D85T, 72 R81K,T(82A)S, N69P M(100C)L

The antibodies listed in Table 8 were analyzed for protein expressionand binding to CD38 via surface plasmon resonance (SPR). Potency assayswere also performed using cell culture supernatant taken fromtransfected cells to assess the relative activity of each of theseanti-CD38-attenuated IFN fusion proteins as outlined in Example 5. Thedata obtained is given in Table 9.

TABLE 9 CD38 Annexin V Caspase binding Assay Assay Anti- by (Fold (FoldCD38- SPR change change Cell attenuated Protein A (RU) relative torelative to Proliferation IFN fusion HPLC at untreated untreated Assayprotein (mg/L) 350 sec* cells) cells) IC₅₀ (μM) A02.2 30.2 756 N/T N/TN/T A02.3 21.6 835 N/T N/T N/T A02.4 27.3 809 N/T N/T N/T A02.5 22.0 788N/T N/T N/T A02.6 33.7 895 N/T N/T N/T A02.12 25.3 N/A 2.7 7.0 236A02.46 3.1 914 2.2 5.8 1190 A02.47 26.5 1455 3.10 4.44 N/T A02.48 3.4921 2.0 5.6 562 A02.49 3.0 946 2.1 5.5 875 A02.50 3.1 809 1.9 6.1 1681A02.51 1.8 368 2.0 5.8 3741 Note: The CD38 binding by SPR refers to theamount of CD38 that remains bound to the surface after 350 seconds ofthe dissociation phase. Annexin V Assay refers to cells positivelystained by Annexin V-FITC after 24 h treatment with antibody constructsat 20 nM. Caspase Assay refers to caspase activation of cells after 24 htreatment with antibody constructs at 20 nM. N/A—Not Available; N/T—NotTested

Of the proteins tested A02.12 expressed well and demonstrated potency inthe Annexin V, Caspase and cell proliferation assays. Substitution ofN69T in antibody A02.47 did not affect expression levels or potency inAnnexin V or Caspase Assays suggesting that removal of this deamidationsite is possible. Substitution N69T could be incorporated into otherconstructs herein to remove this putative deamidation site with minimallosses in the functional activity of the resulting antibody.

Example 2 In Silico Immunogenicity Analysis of the A02.1 Light ChainAmino Acid Sequence

Putative immunogenic epitopes were identified in the light chainvariable region amino acid sequence of A02.1 using the Epibase analysissoftware (Lonza, UK). To remove putative immunogenic epitopes,substitutions were introduced into the A02.1 variable light chain (FIG.4). Light chains with lower predicted immunogenicity were co-expressedin HEK293E cells with the A02.12 heavy chain variable region (SEQ ID NO:34) formatted onto an IgG4 constant region containing the substitutionS228P fused to A145D-attenuated IFN. The antibody variants produced aredetailed in Table 10.

TABLE 10 Variable Variable Anti- Heavy Light CD38- Chain Amino ChainAmino attenuated Acid Variable Acid Variable IFN Substitution HeavySubstitution Light fusion (Relative to SEQ (Relative to SEQ proteinA02.1) ID NO: A02.1) ID NO: A02.18 L74S, I78F, 34 E83I, D85T, 73 R81K,T(82A)S, L47E M(100C)L A02.19 L74S, I78F, 34 E83I, D85T, 74 R81K,T(82A)S, L47G M(100C)L A02.20 L74S, I78F, 34 E83I, D85T, 75 R81K,T(82A)S, L47N M(100C)L A02.21 L74S, I78F, 34 E83I, D85T, 76 R81K,T(82A)S, L47P M(100C)L A02.22 L74S, I78F, 34 E83I, D85T, 77 R81K,T(82A)S, L47S M(100C)L A02.23 L74S, I78F, 34 E83I, D85T, 78 R81K,T(82A)S, L48E M(100C)L A02.24 L74S, I78F, 34 E83I, D85T, 79 R81K,T(82A)S, L48P M(100C)L A02.25 L74S, I78F, 34 E83I, D85T, 80 R81K,T(82A)S, Y49E M(100C)L A02.26 L74S, I78F, 34 E83I, D85T, 81 R81K,T(82A)S, Y49Q M(100C)L A02.27 L74S, I78F, 34 E83I, D85T, 82 R81K,T(82A)S, Y50P M(100C)L A02.28 L74S, I78F, 34 E83I, D85T, 83 R81K,T(82A)S, Y50N M(100C)L A02.29 L74S, I78F, 34 E83I, D85T, 84 R81K,T(82A)S, Y50T M(100C)L A02.30 L74S, I78F, 34 E83I, D85T, 85 R81K,T(82A)S, Y51D M(100C)L A02.31 L74S, I78F, 34 E83I, D85T, 86 R81K,T(82A)S, S52E M(100C)L A02.32 L74S, I78F, 34 E83I, D85T, 87 R81K,T(82A)S, S52H M(100C)L A02.33 L74S, I78F, 34 E83I, D85T, 88 R81K,T(82A)S, S52Q M(100C)L A02.34 L74S, I78F, 34 E83I, D85T, 89 R81K,T(82A)S, H(54A)N M(100C)L A02.35 L74S, I78F, 34 E83I, D85T, 90 R81K,T(82A)S, H(54A)P M(100C)L A02.36 L74S, I78F, 34 E83I, D85T, 91 R81K,T(82A)S, K(54B)D M(100C)L

The above antibodies were analyzed for protein expression, binding toCD38 via SPR, and potency using the cell culture supernatant screen asdescribed in Example 5. The results of these assays are detailed inTable 11. These data indicate that substitution of some residues tolower the predicted immunogenicity of the antibody results inAnti-CD38-attenuated IFN fusion proteins that express and havefunctional potency in the Annexin V, Caspase and Cell ProliferationAssays.

TABLE 11 Annexin Caspase CD38 V Assay Assay binding (Fold (FoldAnti-CD38- Protein by SPR change change Cell attenuated A (RU) atrelative to relative to Proliferation IFN fusion HPLC 350 untreateduntreated Assay protein (mg/L) sec* cells) cells) IC₅₀ (pm) A02.18 9.3745 N/T N/T N/T A02.19 8.0 741 N/T N/T N/T A02.20 9.1 N/T 2.2 5.6 37A02.21 3.3 DNB N/T N/T N/T A02.22 10.4 738 N/T N/T N/T A02.23 15.9 1922.7 6.9 N/T A02.24 23.5 87 1.4 2.3 N/T A02.25 25.7 80 3.0 4.3 2477A02.26 35.0 383 3.2 6.0 66 A02.27 12.3 DNB 1.3 2.5 29910 A02.28 16.1 4222.7 6.3 N/T A02.29 19.7 150 2.7 5.8 133 A02.30 25.2 122 1.9 2.5 N/TA02.31 28.4 359 3.0 7.1 514 A02.32 13.5 663 2.7 7.7 60 A02.33 11.2 107N/T N/T N/T A02.34 16.6 407 4.8 5.1 503 A02.35 11.2 738 2.4 4.8 3050A02.36 16.7 192 2.8 8.0 N/T Note: The CD38 binding by SPR refers to theamount of CD38 that remains bound to the surface after 350 seconds ofthe dissociation phase. Annexin V Assay refers to cells positivelystained by Annexin V-FITC after 24 h treatment with antibody constructsat 20 nM. Caspase Assay refers to caspase activation of cells after 24 htreatment with antibody constructs at 20 nM. DNB—Did not bind; N/T—NotTested

Example 3 Multiple Amino Acid Substitutions Yield Optimized A02.1Variants

By combining substitutions that improve the immunogenicity,manufacturability or potency of the anti-CD38 antibodies described aboveinto a single gene construct, highly optimized anti-CD38 antibodies andanti-CD38-attenuated IFN fusion proteins were obtained. Table 12summarizes such combinatorial substitutions and details heavy- and lightchain combinations co-expressed in HEK293E cells and subsequentlytested.

TABLE 12 Anti- Variable Heavy Variable Light CD38- Chain Amino ChainAmino attenuated Acid Variable Acid Variable IFN Substitution HeavySubstitution Light fusion (Relative to SEQ ID (Relative to SEQ IDprotein A02.1) NO: A02.1) NO: A02.16 L74S, I78F, 34 R29G, Y30S 92 R81K,T(82A)S, E83I, D85T, M(100C)L M89L, N94Q A02.17 L74S, I78F, 34 R29G,Y30S 93 R81K, T(82A)S, E83I, D85T, M(100C)L M89L, N94E A02.52 L74S,I78F, 34 R29G, Y30S, 94 R81K, T(82A)S, S52Q, E83I, M(100C)L D85T, M89L,N94E A02.53 L74S, I78F, 34 Y30S, S52Q, 95 R81K, T(82A)S, E83I, D85T,M(100C)L M89L, N94E A02.54 L74S, I78F, 34 R29G, Y30S, 96 R81K, T(82A)S,S52Q, E83I, M(100C)L D85T, M89L, N94Q A02.55 L74S, I78F, 34 Y30S, S52Q,97 R81K, T(82A)S, E83I, D85T, M(100C)L M89L, N94Q A02.56 L74S, I78F, 34R29G, Y30S, 98 R81K, T(82A)S, S52E, M89L, M(100C)L E83I, D85T, N94EA02.57 L74S, I78F, 34 Y30S, S52E, 99 R81K, T(82A)S, E83I, D85T, M(100C)LM89L, N94E A02.58 L74S, I78F, 34 R29G, Y30S, 100 R81K, T(82A)S, S52E,E83I, M(100C)L D85T, M89L, N94Q A02.59 L74S, I78F, 34 Y30S, S52E, 101R81K, T(82A)S, E83I, D85T, M(100C)L M89L, N94Q A02.60 L74S, I78F, 34R29G, Y30S, 102 R81K, T(82A)S, S52Q, N69Q, M(100C)L E83I, D85T, M89L,N94E A02.61 L74S, I78F, 34 Y30S, S52Q, 103 R81K, T(82A)S, N69Q, E83I,M(100C)L D85T, M89L, N94E A02.62 L74S, I78F, 34 R29G, Y30S, 104 R81K,T(82A)S, S52Q, N69Q, M(100C)L E83I, D85T, M89L, N94Q A02.63 L74S, I78F,34 Y30S, S52Q, 105 R81K, T(82A)S, N69Q, E83I, M(100C)L D85T, M89L, N94QA02.64 L74S, I78F, 34 R29G, Y30S, 106 R81K, T(82A)S, S52E, N69Q,M(100C)L E83I, D85T, M89L, N94E A02.65 L74S, I78F, 34 Y30S, S52E, 107R81K, T(82A)S, N69Q, E83I, M(100C)L D85T, M89L, N94E A02.66 L74S, I78F,34 R29G, Y30S, 108 R81K, T(82A)S, S52E, N69Q, M(100C)L E83I, D85T, M89L,N94Q A02.67 L74S, I78F, 34 Y30S, S52E, 109 R81K, T(82A)S, N69Q, E83I,M(100C)L D85T, M89L, N94Q

Each antibody described in Table 12 was analyzed for protein expression,binding to CD38 via SPR, and potency using cell culture supernatant. Theresulting data is given in Table 13. These results demonstrate thatcombining substitutions predicted to be beneficial in silico gave riseto some Anti-CD38-attenuated IFN fusion proteins that expressed and hadfunctional potency in the Annexin V, Caspase and Cell ProliferationAssays.

TABLE 13 CD38 Annexin binding V Assay Caspase Cell Anti- by (Fold Assay(Fold Pro- CD38- Protein SPR change change liferation attenuated A (RU)at relative to relative to Assay IFN fusion HPLC 350 untreated untreatedIC₅₀ protein (mg/L) sec* cells) cells) (pm) A02.14 8.1 1247 2.18 5.47398 A02.15 29.6 1409 3.92 5.62 491 A02.16 5.7 1050 2.4 7.9 636 A02.1710.0 1103 3.7 5.7 467 A02.52 2.8 416 2.0 5.4 2665 A02.53 3.0 545 2.2 4.76338 A02.54 1.8 250 1.9 5.9 15350 A02.55 2.1 436 2.2 4.5 12740 A02.562.0 178 1.7 4.3 13860 A02.57 2.7 345 2.5 6.6 6363 A02.58 2.3 273 1.9 4.99142 A02.59 1.2 388 2.0 4.7 6176 A02.60 1.3 DNB 1.5 3.3 185600 A02.611.2 DNB 1.7 4.1 65160 A02.62 1.3 DNB 1.8 5.0 55590 A02.63 1.2 DNB 1.73.4 152100 A02.64 1.1 DNB 1.8 3.2 89120 A02.65 1.4 DNB 1.6 4.1 37240A02.66 1.3 DNB 1.6 4.0 57540 A02.67 1.6 DNB 2.3 3.9 82760 Note: The CD38binding by SPR refers to the amount of CD38 that remains bound to thesurface after 350 seconds of the dissociation phase. Annexin V Assayrefer to cells positively stained by Annexin V-FITC after 24 h treatmentwith antibody constructs at 20 nM. Caspase Assay refers to caspaseactivation of cells after 24 h treatment with antibody constructs at 20nM DNB—Did not bind; N/T—Not Tested

Example 4 Pairing of Different Heavy and Light Chain Anti-CD38Antibodies

In order to determine if functional anti-CD38-attenuated IFN fusionproteins could be obtained, the heavy (SEQ ID NO: 110) and light (SEQ IDNO: 112) chains from the antibody X910/12-HC-L0-IFN-alpha (A145D) IgG4described in PCT/AU2012/001323, and the heavy (SEQ ID NO: 111) and light(SEQ ID NO: 113) chains from the antibody X913/15-HC-L0-IFN-alpha(A145D) IgG4 described in PCT/AU12/001323, were paired with each otherin various combinations and with heavy and lights chains described inthe foregoing examples. A summary of the heavy and light chain pairingsis provided listed in Table 14.

TABLE 14 Anti-CD38-attenuated Variable Heavy Variable Light IFN fusionprotein SEQ ID NO: SEQ ID NO: A02.38 34 113 A02.39 34 112 A02.40 111 65A02.41 110 65 X913/15-HC-L0-IFN- 111 113 alpha (A145D) IgG4 A02.43 110113 A02.44 111 112 X910/12-HC-L0-IFN- 110 112 alpha (A145D) IgG4

Each antibody produced was analyzed for protein expression, binding toCD38 via SPR, and potency using the cell culture supernatant potencyassays. The results of these assays are presented in Table 15a. Thesedata show that pairing different heavy and light chains from distinctantibodies gave rise to some Anti-CD38-attenuated IFN fusion proteinsthat expressed and had functional potency in the Annexin V, Caspase andCell Proliferation Assays.

TABLE 15a Annexin V Caspase Assay (Fold Assay (Fold Anti-CD38- CD38change change attenuated Protein A binding by relative to relative toIFN fusion HPLC SPR (RU) untreated untreated protein (mg/L) at 350 sec*cells) cells) A02.38 2.9 DNB 0.9 4.3 A02.39 21.3 66 2.5 6.6 A02.40 2.1DNB 1.0 4.4 A02.41 50.0 165 2.6 6.7 X913/15-HC- 8.2 96 2.2 5.4L0-IFN-alpha (A145D) IgG4 A02.43 2.5 DNB 0.9 4.1 A02.44 2.2 DNB 1.0 4.5X910/12-HC- 27.1 125 2.6 5.9 L0-IFN-alpha (A145D) IgG4 Note: The CD38binding by SPR refers to the amount of CD38 that remains bound to thesurface after 350 seconds of the dissociation phase. Annexin V Assayrefer to cells positively stained by Annexin V-FITC after 24 h treatmentwith antibody constructs at 20 nM. Caspase Assay refers to caspaseactivation of cells after 24 h treatment with antibody constructs at 20nM. DNB—Did not bind.

A selection of the above Anti-CD38-attenuated IFN fusion proteins werepurified and analyzed for binding to CD38 positive cells in cell basedassays. In addition potency assays were repeated to give a more accuratedetermination of the relative activity of each Anti-CD38-attenuated IFNfusion protein. The results of these assays are given in Table 15b.

TABLE 15b Annexin Caspase V Assay Assay Cell Anti- ARP-1 (Fold (FoldPro- CD38- Flow change change liferation attenuated binding relative torelative to Assay IFN fusion (EC₅₀, in untreated untreated IC50 proteinμg/mL) cells) cells) (pM) Figures A02.1 1.45 1.86 4.2 278.2 13, 14, 15,16, 17, 18, 19 A02.2 2.46 1.94 4.4 175.7 13, 19 A02.3 0.96 1.86 3.5254.9 13, 19 A02.4 1.50 1.88 3.8 198.3 13, 19 A02.5 1.45 1.88 4.3 146.313, 19 A02.6 1.42 2.03 3.5 102.3 13, 15, 19, 20 A02.8 1.93 1.91 3.8125.5 13, 19 A02.9 1.03 1.96 3.6 107.1 13, 19 A02.10 1.48 1.95 3.8 125.613, 19 A02.11 3.48 1.98 3.9 374 13, 19 A02.12 3.40* 1.40 3.4 23.66 14,16, 19, 21 A02.14 10.01* 1.82 2.5 398.20 19 A02.15 1.97* 3.09 6.8 491.7019 A02.16 3.89* 2.66 5.2 636.80 14, 19 A02.17 9.32* 2.23 3.2 467.1 14,19 A02.18 1.64 1.55 3.7 78.72 19 A02.19 1.07 1.63 3.5 230.3 19 A02.2015.92* 1.61 2.9 36 19 A02.22 1.58 1.91 3.8 207 19 A02.25 0.37* 1.42 2.42477 19 A02.26 37.99* 1.56 2.0 66 19 A02.27 LB* 1.06 1.0 29910 14, 16,19 A02.29 0.48* 1.55 3.0 133 19 A02.31 0.26* 1.78 2.1 514 14, 16, 19A02.32 LB* 1.83 3.3 605 19 A02.33 1.54 1.96 3.7 741 16, 19 A02.34 0.89*3.06 4.7 503 19 A02.35 3.44* 1.71 1.5 3050 19 A02.37 LB* 2.05 4.3 128 19A02.39 LB* 1.92 4.3 3714 19 A02.41 0.78* 2.01 3.4 554 14, 19 A02.42 LB*1.52 3.3 310 19 A02.45 0.71* 1.66 1.6 1697 19 A02.47 16.35* 1.53 4.69144.3 19 The flow binding refers to the concentration of antibodyrequired to achieve 50% of maximal mean fluorescence intensity. AnnexinV Assay refer to cells positively stained by Annexin V-FITC after 24 htreatment with antibody constructs at 20 nM. Caspase Assay refers tocaspase activation of cells after 24 h treatment with antibodyconstructs at 20 nM. LB—low binding, not sufficient for an EC₅₀ value.*Antibody was tested in a flow binding assay against H929 cell line.Reported value is the EC₅₀ in μg/mL.

FIG. 5 lists the consensus variable heavy chain and FIG. 6 lists theconsensus variable light chain of A02.1 related constructs withfunctional activity. It could be further envisioned that combinations ofsubstitutions could be made such as those described for Anti-CD38antibodies X02.114, X02.115, X02.116, X02.117, X02.118, X02.119 (FIG.6), X02.120, X02.121, X02.122, X02.123, X02.124, X02.125, X02.126 orX02.127 (FIG. 30). Further the above Anti-CD38 antibodies could also beconstructed as Anti-CD38-attenuated IFN fusion proteins and tested forfunctional activity as described herein.

H929 Multiple Myeloma Xenograft Model

The in vivo potency of A02.1 has been tested previously in the NCI-H929s.c. multiple myeloma model as described in Example 5. A02.1 was shownto have potent anti-tumor activity. The data is presented inPCT/AU2012/001323.

The H929 multiple myeloma xenograft model could be used to test theanti-tumor activity of any of the Anti-CD38-attenuated IFN fusionproteins described above.

Attenuated IFN is Required for Potent Apoptotic and Caspase Activationin Tumor Cell Lines

Using the Annexin V assay and the Caspase Assay it was demonstrated thatthe potent apoptotic activity and caspase activation is dependent on theAnti-CD38-attenuated IFN fusion proteins containing an attenuated IFN(Table 16a, FIG. 18). In the Annexin V Assay the attenuated IFNcontaining proteins (A02.1 and A02.6) had 2-fold greater activity thanthe proteins not containing attenuated IFN (X02.1 and X02.6).

TABLE 16a Annexin V Caspase Anti- Assay (Fold Assay (Fold CD38- changechange attenuated relative to relative to IFN fusion untreated untreatedprotein cells) cells) A02.1 3.57 5.60 X02.1 1.50 2.34 A02.6 2.03 3.5X02.6 1.04 0.40 Annexin V Assay refer to cells positively stained byAnnexin V-FITC after 24 h treatment with antibody constructs at 20 nM.Caspase Assay refers to caspase activation of cells after 24 h treatmentwith antibody constructs at 20 nM.

Example 5 General Methods

Production of antibodies and antibody-fusion constructs in HEK-293Ecells. DNA plasmids encoding protein constructs (antibodies andantibody-IFN-alpha2b related constructs) were prepared using HiSpeedPlasmid Maxi Kit (Qiagen, Valencia, Calif.) and then transfected intoHEK293E cells (CNRC, Montreal, Canada), grown in F17 synthetic mediumsupplemented with 0.45% (w/v) D-(+)-Glucose (Sigma, Castle Hill, NSW),25 μg/mL Geneticin (Invitrogen, Carlsbad, Calif.), and 1×GlutaMAX(Invitrogen, Carlsbad, Calif.) using a commercially availabletransfection reagent and OptiMEM medium (Invitrogen, Carlsbad, Calif.).After allowing for expression for 6 days in an incubator supplied with5% CO₂ and 120 rpm shaking, the culture media was isolated and subjectedto affinity purification using Protein A Mab Select SuRe™ agarose beads(GE Healthcare, Piscataway, N.J.). Purified protein constructs werebuffer-exchanged into 0.2M arginine HCl, 25 mM citric acid, 71.5 mMsodium hydroxide at pH 6.0 using a PD Midi-Trap G-25 column (GEHealthcare, Piscataway, N.J.) or a HiPrep 26/10 Desalting column (HiTrapDesalting HiPrep 26/10 Desalting). Purified protein constructs were thenconcentrated using 50 kDaAmicon Ultra centrifugal filter devices(Millipore, Billerica, Mass.), followed by protein concentrationdetermination by reading absorbance at 280 nm.

Production of Antibodies and Antibody-Fusion Constructs in CHO Cells.

DNA plasmids encoding protein constructs (antibodies andantibody-IFN-alpha2b related constructs) were prepared using HiSpeedPlasmid Maxi Kit (Qiagen, Valencia, Calif.) and then transfected intoCHO cells (Lonza) grown in Freestyle™ CHO Expression Medium (Invitrogen,Carlsbad, Calif.) using a commercially available transfection reagentand OptiPro SFM™ medium (Invitrogen, Carlsbad, Calif.). After allowingfor expression for 6 days in an incubator supplied with 10% CO₂ and 120rpm shaking, the culture media was isolated and subjected to affinitypurification using Protein A Mab Select SuReagarose beads (GEHealthcare, Piscataway, N.J.). Purified protein constructs werebuffer-exchanged into 0.2M arginine.HCl, 25 mM citric acid, 71.5 mMsodium hydroxide at pH 6.0 using a PD Midi-Trap G-25 column (GEHealthcare, Piscataway, N.J.) or a HiPrep 26/10 Desalting column (HiTrapDesalting HiPrep 26/10 Desalting). Purified protein constructs were thenconcentrated using 50 kDaAmicon Ultra centrifugal filter devices(Millipore, Billerica, Mass.), followed by protein concentrationdetermination by reading absorbance at 280 nm.

Anti-CD38-attenuated IFN fusion proteins binding to CD38 as measured bySurface Plasmon Resonance (SPR). The capacity of anti-CD38 antibodiesand anti-CD38-attenuated IFN fusion proteins to bind to human CD38 weremeasured using unpurified transfected cell supernatant prepared 7:1 withNon Specific Binding Reducer (GE Healthcare, Piscataway, N.J.). Briefly,using a Biacore™ 3000 or a T200, Protein A was immobilized onto FlowCell (FC) 1 (FC1) and FC2 (or alternatively FC3 and FC4) of a CM5research grade sensor chip using amine coupling, giving approximately1500 RU. FC2 (or FC4) was used as a reference throughout theexperiments. The experiments were run at 37° C. in HBS-P+ buffer (0.01 MHEPES, 0.15 M NaCl, 0.005% v/v Surfactant P20, pH 7.4). At a flow rateof 20 l/min, both flow cells were regenerated with 10 μL 50 mM sodiumhydroxide before 40 μL supernatant containing the protein was passedover FC1 (or FC3) only. 301 μL of CD38 (10 g/mL in running buffer) or 30μL running buffer was injected over FC1 and FC2 with a 5 minutedissociation time. Both surfaces were regenerated twice with sodiumhydroxide. Results were generated using the BIAevaluation softwareprovided with the machine. Microsoft Excel was used for calculations.BIAevaluation software automatically subtracted the reference sensorgramgiving a trace of FC2-1 (or FC4-3) for each sample. A double referencewas performed for each antibody tested by subtracting the sensorgramwith a CD38 injection from the sensorgram with a blank running bufferinjection. The Protein A capture refers to the response units measuredfrom a sensorgram at a fixed timepoint of 412.5 s and this correspondsto the level of protein captured on the Protein A surface. CD38 bindingis the response units measured at 507.5 s and is an indication of thelevel of bound CD38 to the protein captured sensor. CD38 dissociation isthe response units measured at 865.5 s and is an indication of the levelof CD38 bound to the protein captured surface after approximately 300 sof dissociation phase. BIAevalution was used to fit the sensorgram usinga Langmuir 1:1 equation in order to generate an equilibrium associationconstant (KD)

Protein A HPLC.

Supernatants were analyzed by Protein A HPLC using a POROS A/20 2.1×30mm Id column (Applied Biosystems) connected to an Agilent 1100chromatography system. The column was equilibrated with PBS pH7.4, andprotein was eluted with PBS adjusted to pH 2.2. A standard curve wasgenerated using known amounts of a monoclonal antibody in PBS. Thechromatograms, at the wavelengths of 215 nm or 280 nm, were integratedusing the manufacturer's software and the area under the curve (AUC)reported and interpolated against the generated standard curve toestimate concentration.

Flow Cytometry Binding of Antibodies and Anti-CD38-Attenuated IFN FusionProteins to a Human CD38 Positive Cell Line, ARP-1 and H929.

The multiple myeloma cell line ARP-1 was a gift from Bart Barlogie MD,PhD, Director of the Myeloma Institute at the University of ArkansasMedical Center (Little Rock, Ak.). It is described in Hardin J. et al.(1994) Blood. 84:3063-70). The multiple myeloma cell line NCI-H929(H929) was purchased from ATCC (CRL-9068, Gazdar, Blood 67: 1542-1549,1986).

The ability of the antibodies or antibody-interferon constructs to bindthe human CD38-positive myeloma cell lines ARP-1 or H929 in flowcytometry-based assays was tested. ARP-1 cells or H929 cells (5×10⁵, asjudged by trypan blue exclusion) were incubated with each protein orwith a human IgG4 monoclonal antibody with irrelevant specificityprotein construct at various concentrations in 50 μL of FACS buffer (PBSplus 1% fetal calf serum, FCS, 0.2M HEPES, 0.5M EDTA) in 96 well platesfor 60 minutes on ice in the dark. Cells were washed three times withFACS buffer before incubation for 30 minutes in 50 μL of FACS buffercontaining goat anti-human IgG (Fc-specific, conjugated to fluoresceinisothiocyanate, FITC; Sigma-Aldrich, St. Louis, Mo.). After washingthree times with FACS buffer, cells were fixed with 50 μL of PBScontaining 4% formaldehyde v/v and incubated at 4° C. in the dark for 16hours. Incubated cells in suspension were diluted with an additional 150μL of FACS buffer and analyzed for binding by flow cytometry on a FACSCanto II (BD Biosciences, San Diego, Calif.) using forward scatter, sidescatter and fluorescence intensity in the FITC channel. The valuereported is the mean fluorescence intensity (MFI).

Target Assays

Daudi cell proliferation assay: This assay was used to quantify theanti-proliferative activity of IFNs and antibody-IFN fusion proteinconstructs on cells that display CD38. Daudi cells express CD38 as acell surface associated antigen. The viability of cells was measuredusing the reagent CellTiter-Glo®, Cat #G7570, from Promega (Madison,Wis.). This is a luminescence-based assay that determines the viabilityof cells in culture based on quantitation of ATP. The signal strength isproportional to the number of viable cells in a microtiter plate well.The details of the assay are as follows: Daudi cells (obtained fromATCC, Manassas, Va.) were cultured in a T75 flask (TPP, Trasadingen,Switzerland, cat#90076) to a preferred density of between 0.5×10⁵ and0.8×10⁵ viable cells/mL in RPMI 1640 (Mediatech, Inc., Manassas, Va.,cat #10-040-CV) with 10% Fetal Bovine Serum (FBS; Hyclone, Logan, Utahcat# SH30070.03). Cells were harvested by centrifuging at 400×g for fiveminutes, decanting the supernatant, and resuspending the cell pellet inRPMI 1640+10% FBS. Cells were then counted and the density was adjustedto 3.0×10⁵ cells/mL in RPMI 1640+10% FBS. Then, 501 μL of cellsuspension was aliquoted into each well of a 96 well round bottom tissueculture plate (hereafter, “experimental plate”) (TPP, cat#92067). On aseparate, sterile 96 well plate (hereafter, “dilution plate”; Costar,Corning, N.Y. cat#3879), test articles were serially diluted induplicate in RPMI 1640+10% FBS. Then, 50 μL/well was transferred fromthe dilution plate to the experimental plate. The experimental plate wasthen incubated for four days at 37° C. with 5% CO₂. A mixture of themanufacturer-supplied assay buffer and assay substrate (hereafter,“CellTiter-Glo® reagent”, mixed according to the manufacturer'sinstructions) was added to the experimental plate at 100 μL/well. Theplate was shaken for two minutes.

Then, 100 μL/well was transferred from the experimental plate to a 96well flat bottom white opaque plate (hereafter, “assay plate”; BDBiosciences, Franklin 5 Lakes, N.J. cat#35 3296). The content of theassay plate was then allowed to stabilize in the dark for 15 minutes atroom temperature. The plate was read on a Victor 3V Multilabel Counter(Perkin Elmer, Waltham, Mass., model#1420-041) on the luminometrychannel and the luminescence was measured. Results are presented as“relative luminescence units” (RLU). Data was analyzed using Prism 5(Graphpad, San Diego, Calif.) using non-linear regression and threeparameter curve fit to determine the midpoint of the curve (EC50).

ARP-1 Cell proliferation assay. This assay was used to quantify theanti-proliferative activity of IFNs and antibody-IFN fusion proteinconstructs against CD38 antigen positive cells. ARP1 cells express CD38as cell surface associated antigens. The viability of cells was measuredusing the reagent CellTiter-Glo®, Cat #G7570, from Promega (Madison,Wis.). This is a luminescence-based assay that determines the viabilityof cells in culture by quantitation of ATP. The signal strength isproportional to the number of viable cells in a microtiter plate well.

The details of the assay are as follows: ARP-1 cells were cultured in aT175 flask (Costar, Corning, N.Y. Lakes, N.J., cat# CLS431080) to apreferred density of between 2.0×10⁵ and 2.0×10⁶ viable cells/mL in RPMI1640 (Life Technologies, Mulgrave, VIC, cat #11875-093) with 10% FetalBovine Serum (FBS; AusGeneX, Molendinar, QLD, Australia cat#FBS500S).Cells were harvested by centrifuging at 400×g for five minutes,decanting the supernatant, and resuspending the cell pellet in RPMI1640+10% FBS. Cells were then counted and the density was adjusted to2.0×10⁵ cells/mL in RPMI 1640+10% FBS. Then, 50 μL of the cellsuspension was aliquoted into each well of a 96-well flat bottom whiteopaque plate (hereafter, “experimental plate”; Costar, Corning, N.Y.Lakes, N.J., cat# CLS3917). On a separate, sterile 96-well plate(hereafter, “dilution plate”; Costar, Corning, N.Y. cat#3799), testarticles were serially diluted in duplicate in RPMI 1640+10% FBS.Subsequently, 50 μL/well was transferred from the dilution plate to theexperimental plate. The experimental plate was then incubated for fourdays at 37° C. with 5% CO₂. Each experimental plate included theparental antibody IFN construct as the relative control.

A mixture of the manufacturer-supplied assay buffer and assay substrate(CellTiter-Glo® reagent, mixed according to the manufacturer'sinstructions) was added to the experimental plate at 100 μL/well. Theplate was shaken for two minutes. The content of the assay plate wasthen allowed to stabilize in the dark for 15 minutes at roomtemperature. The plate was read on a FLUOstar Galaxy plate reader (BMGLabtech, Durham, N.C.) on the luminometry channel and the luminescencewas measured. Data was analyzed using Prism 5 (Graphpad, San Diego,Calif.) using non-linear regression and three parameter curve fit todetermine the midpoint of the curve (EC50).

Annexin V assay. H929 cells were harvested by centrifuging at 400×g forfive minutes, decanting the supernatant, and resuspending the cellpellet in RPMI 1640+10% FBS. Cells were then counted and the density wasadjusted to 1.0×10⁶ cells/mL in RPMI 1640+10% FBS. Then, 50 μL of thecell suspension was aliquoted into each well of 96-well round bottomclear plates (hereafter, “experimental plate;” Costar, Corning, N.Y.cat#CL3799). On a separate, sterile 96-well plate (hereafter, “dilutionplate”; Costar, Corning, N.Y. cat# CL3799), test articles were dilutedto 40 nM in quaduplicate in RPMI 1640+10% FBS. Subsequently, 50 μL/wellwas transferred from the dilution plate to the experimental plate. Theexperimental plate was then incubated for 24 hours at 37° C. with 5%CO₂. The cells were then centrifuged at 400×g for 5 min, supernatantdecanted and resuspended in 100 μL of HEPES buffer containing AnnexinV-FITC (1/200) and 7-AAD (1/50). The cells were then incubated for 15min at room temperature and subsequently analyzed for Annexin V and7-AAD staining by flow cytometry on a FACS Canto II (BD Biosciences, SanDiego, Calif.) using forward scatter, side scatter, FITC and PerCP-Cy5.5channels. Annexin V positive cells refer to cells positively stained byAnnexin V-FITC after 24 h treatment with antibody constructs at 20 nMand is expressed as fold change relative to untreated cells.

Caspase assay. Activated caspases 2, 3, 6, 7, 8, 9, 10 were measuredwith the reagent Homogeneous Caspases Assay, fluorimetric Cat#12236869001, from Roche (West Sussex, UK) after treatment with testantibodies. The details of the assay follow.

ARP-1 cells, which express high levels of CD38, were cultured in a T175flask (Costar, Corning, N.Y., cat# CLS431080) to a preferred density ofbetween 2.0×10⁵ and 2.0×10⁶ viable cells/mL in RPMI 1640 (LifeTechnologies, Mulgrave, VIC, cat #11875-093) with 10% FBS (AusGeneX,Molendinar, QLD, Australia cat# FBS500S). Cells were harvested bycentrifuging at 400×g for five minutes, decanting the supernatant, andresuspending the cell pellet in RPMI 1640 Phenol red-free (LifeTechnologies, Mulgrave, VIC, cat #11835-030) +10% FBS. Cells were thencounted and the density was adjusted to 2.0×10⁵ cells/mL in RPMI 1640Phenol red free+10% FBS. Then, 50 μL of the cell suspension wasaliquoted into each well of a 96-well flat bottom black-walled clearbottom plate (hereafter, “experimental plate”; Costar, Corning, N.Y.cat# CLS3603). On a separate, sterile 96-well plate (hereafter,“dilution plate”; Costar, Corning, N.Y. cat#3799), test articles werediluted to 40 nM in quadruplicate in RPMI 1640 Phenol red free+10% FBS.Subsequently, 50 μL/well was transferred from the dilution plate to theexperimental plate. The experimental plate was then incubated for 24hours at 37° C. with 5% CO₂. The manufacturer-supplied assay buffer wasadded to the manufacturer-supplied substrate and mixed according to themanufacturer's instructions to create the “substrate solution.” Then,100 μL of the substrate solution was added to each well of the assayplate. The plate was shaken for 2 minutes. The plate was then incubatedat room temperature for 15 minutes in the dark and finally read onFluoStar Galaxy plate reader (BMG Labtech, Durham, N.C.) with anexcitation filter 470-500 nm and emission filter 500-560 nm and thefluorescence measured and presented as fold change relative to untreatedcells. Caspase Assay refers to caspase activation of cells after 24 htreatment with antibody constructs at 20 nM.

Off-Target Assays

iLite gene reporter assay. The “off-target” iLite assay (PBL InterferonSource, Piscataway, N.J., Cat#51100) was performed largely as describedby the manufacturer, with the addition of a human IgG blocking step. TheiLite cell line is described by the manufacturer as “a stabletransfected cell line derived from a commercially availablepro-monocytic human cell line characterized by the expression of MHCClass II antigens, in particular the human lymphocyte antigen (HLADR),on the cell surface.” The cell line contains a stably transfectedluciferase gene, the expression of which is driven by aninterferon-response element (IRE), which allows for interferon activityto be quantified based on luminescence output. The manufacturer suppliediLite plate (hereafter, assay plate) and diluent were removed from the−80° C. freezer and allowed to equilibrate to room temperature. Then, 50μL of the diluent was added per well to the assay plate. The vial ofmanufacturer-supplied reporter cells was removed from the −80° C.freezer and thawed in a 37° C. water bath. Then, 25 μL aliquots of cellswere dispensed into each well of the assay plate. Next, 12.5 μL of 8mg/mL human IgG that was diluted into RPMI 1640+10% FBS (SigmaChemicals, St. Louis, Mo.; cat#14506) was added per well. The contentswere mixed and incubated at 37° C. for 15 minutes. On a separate“dilution plate,” test articles were serially diluted in duplicate inRPMI 1640+10% FBS. Then, 12.5 μL of the test articles were transferredfrom the dilution plate to the assay plate. The assay plate was thenincubated at 37° C. with 5% CO₂ for 17 hours. The manufacturer-suppliedassay buffer and substrate were removed from the −80° C. freezer andallowed to equilibrate to room temperature for two hours. Themanufacturer-supplied assay buffer was added to themanufacturer-supplied substrate vial and mixed well according to themanufacturer's instructions to create the “luminescence solution.” Then,100 μL of the luminescence solution was added to each well of the assayplate. The plate was shaken for 2 minutes. The plate was then incubatedat room temperature for 5 minutes in the dark and finally read on aVictor 3V Multilabel Counter on a luminometry channel and theluminescence measured and presented as RLU. The data was analyzed withGraphpad Prism 5 as described for the “on-target (Daudi) assay.” To testanti-CD38 antibody-IFN fusion protein constructs in the iLite assay,manufacturer-supplied diluent was supplemented with 0.25 mg/mL anti-CD38antibody (same antibody clone being tested as an antibody-IFN fusionprotein construct, to block any binding of the anti-CD38 antibody-IFNfusion protein constructs to the CD38 expressed on the iLite cells).

HEK-Blue Off-target assay. The assay was used to quantify the ability ofantibody-IFN fusion constructs to bind interferon-alpha/β receptor(IFNAR) using the HEK-Blue™ IFN-alpha/β cell line (InvivoGen, San Diego,Calif.). The “off-target (HB-IFN) assay” was performed largely asdescribed by the manufacturer of the HEK-Blue IFN-alpha/β cell line.HEK-Blue™ IFN-alpha/β Cells are specifically designed to monitor theactivation of the JAK-STAT pathway, which is induced by type I IFNs. Thecells were generated by introducing the human STAT2 and IRF9 genes intoHEK293 cells to obtain a fully active type I IFN signalling pathway. TheHEK-Blue™ IFN-alpha/β Cells stably express a reporter gene, secretedembryonic alkaline phosphatase (SEAP), under the control of the ISG54promoter. ISG54 is a well-known ISG activated through an ISRE-dependentmechanism by type I IFNs. Upon IFN-alpha or IFNβ stimulation, HEK-Blue™IFN-alpha/3 cells activate the JAK-STAT pathway and then the expressionof the SEAP reporter gene. SEAP is secreted into the media and can bequantitated using the colorimetric reagent QUANTI-Blue™. Briefly,HEK-Blue IFN-alpha/3 cells (Invivogen, San Diego Calif. cat# hkb-ifnab)were thawed and cultured in DMEM media (Mediatech, Manassas Va.,cat#10-013-CV)+10% FBS (Hyclone, Logan Utah, cat# SH30070.03) that hadbeen heat inactivated (HI FBS). When the cells reached 60-80%confluence, they were lifted with Cell Stripper (Mediatech,cat#25-056-CI). Cells were washed twice in DMEM+HI FBS and counted.Cells were adjusted to 3.3×10⁵ viable cells/mL in DMEM+HI FBS and 150 μLwas aliquoted per well into a flat bottom 96 well tissue culture plate(hereafter, the “experimental plate”). Then, 50 μL of IFN-alpha2b orfusion protein construct, diluted into DMEM+HI FBS, was added per well.The plate was incubated at 37° C. 5% CO₂ for 16-24 hours. QUANTI-Blue(Invivogen, cat# rep-qbl) was prepared according to the manufacturer'sdirections. QUANTI-Blue (150 μL) was aliquoted into each well of a flatbottom plate (hereafter, the “assay plate”). Then, 50 μL supernatant perwell from the experimental plate was transferred to assay plate. Assayplate was then incubated at 37° C. for 1-3 hours. Assay plate absorbanceat 630 nm was read on a model 1420-41 Victor 3V Multilabel Counter fromPerkin-Elmer. Data was analyzed using Graph Pad Prism.

H929 Xenograft Model

The effect of different doses of the A10.38 and A10.0anti-CD38-attenuated IFN-alpha fusion protein constructs, were comparedto the non-CD38-targeted fusion protein construct, on myeloma tumorgrowth. For these comparisons, the NCI-H929 s.c. multiple myeloma modelwas used.

The multiple myeloma cell line, NCI-H929 (ATCC CRL-9068, Gazdar, Blood67: 1542-1549, 1986) is grown subcutaneously in immunocompromised (SCID)mice.

Eight to twelve week old CB.17 SCID mice were injected subcutaneously inthe flank with 1×10⁷ NCI-H929 tumor cells in 50% Matrigel. When averagetumor size reached 170-350 mm³, mice were grouped into 4 cohorts of 7mice each and treatment began at time zero (TO). All treatments weregiven by intraperitoneal injection, (i.p.) twice weekly for 3 weeks(indicated by bar under graph). All compounds were dosed at 100 μg/dose(approximately 4.5 mg/kg) except vehicle group. Tumor volume wasmeasured twice weekly by caliper measurement. Endpoint was tumor volumeof 2,000 mm³.

The effect of different doses of the A02. 6, A10.0 and A10.38anti-CD38-attenuated IFN-alpha fusion protein constructs, were comparedto vehicle, on myeloma tumor growth. For these comparisons, the NCI-H929s.c. multiple myeloma model was used.

The multiple myeloma cell line, NCI-H929 (ATCC CRL-9068, Gazdar, Blood67: 1542-1549, 1986) is grown subcutaneously in immunocompromised (SCID)mice.

Eight to twelve week old CB.17 SCID mice were injected subcutaneously inthe flank with 1×10⁷ NCI-H929 tumor cells in 50% Matrigel. When averagetumor size reaches 90 mm³, mice will be grouped into 4 cohorts of 5 miceeach and treatment begin at time zero (TO). All treatments will be givenby intraperitoneal injection, (i.p.) twice weekly for 3 weeks (indicatedby bar under graph). All compounds will be dosed at 100 μg/dose(approximately 4.5 mg/kg) except vehicle group. Tumor volume will bemeasured twice weekly by caliper measurement.

Example 6 Anti-CD38-Attenuated IFN Fusion Protein with AlternativeConstant Region

A02.12 comprises an anti-CD38-attenuated IFN fusion protein in which theconstant region of the protein is HC-L0-IFN-alpha (A145D) IgG4 (SEQ IDNO: 9). The heavy chain variable region of this antibody was reformattedonto an IgG1 constant region fused to A145D attenuated IFN-alpha2b (SEQID NO: 10). Co-expression of this heavy chain with the light chain ofX02.107 (SEQ ID NO: 65) in HEK293E cells yielded antibody A02.112.Comparison of antibodies A02.12 and A02.112 using flow cytometry-basedCD38-binding assays and potency assays demonstrates that other antibodyconstant regions, such as human IgG1, may also be used resulting inantibody-attenuated IFN fusion proteins with potent biologic activityequivalent to those generated using a human IgG4 constant region (Table16b).

TABLE 16b Annexin Caspase V Assay Assay Cell Anti- Flow (Fold (Fold Pro-CD38- binding change change liferation attenuated (EC₅₀ relative torelative to Assay IFN fusion in μg/ untreated untreated IC₅₀ protein mL)cells) cells) (pm) Figures A02.12 3.40* 1.40 2.70 23.66 FIG. 21 A02.112<0.3*   3.14 3.74 N/T FIG. 21 Note: *Antibody was tested in a flowbinding assay against H929 cell line. Reported value is the EC₅₀ inμg/mL. Annexin V Assay refer to cells positively stained by AnnexinV-FITC after 24 h treatment with antibody constructs at 20 nM. CaspaseAssay refers to caspase activation of cells after 24 h treatment withantibody constructs at 20 nM. N/T—Not Tested

Example 7 Humanization of R5D1, R5E8 and R10A2 Variable Regions

Rat-derived anti-CD38 antibodies R5D1, R5E8 and R10A2 are described inPCT/AU12/001323 and were selected for humanization. The variable regionsof these antibodies were superhumanized as described in U.S. Publ. No.2003/0039649. Briefly, canonical structures were assigned to each rodentheavy and light chain through inspection of their respective amino acidsequences. R10A2 was assigned the canonical structure 2-1-1/1-2(V_(L)JV_(H)), R5E8 was assigned the canonical structure 4-1-1/1-2, andR5D1 was assigned the canonical structure 2-1-1/1-2. Human germlinesequences of the same canonical structure were used as acceptorframeworks for the grafting of donor CDRs. Variants of the resultingsuperhumanized antibody genes containing amino acid substitutions atpositions within their sequences deemed likely to be important formaintenance of their binding activity were also designed. The differentheavy chain superhumanized variable regions are shown in FIG. 7. Thedifferent light chain superhumanized variable regions are shown in FIG.8.

Heavy chain variable region sequences were subcloned into vector pEE6.4containing a human IgG4 constant region possessing the substitutionS228P fused to A145D attenuated IFN-alpha2b (SEQ ID NO: 9). Light chainvariable regions were subcloned into vector pEE12.4 containing a humankappa constant region (SEQ ID NO: 5). Antibodies were produced throughco-expression of heavy chains in pEE6.4 and light chains in pEE12.4 inCHO cells as described previously. Table 17 summarises the heavy- andlight chain pairings used to produce each superhumanized 5D1-basedprotein. Table 18 details the heavy- and light chain pairings for thesuperhumanized 5E8-based protein generated, whilst the heavy- and lightchain pairings used to generate superhumanzied 10A2-based proteins aregiven in Table 19. One-shot equilibrium dissociation constant (K_(D))ranking of the superhumanized antibodies was performed by BIAcore™analysis of the resulting CHO transfection supernatants. The method wasused to determine if the antibodies expressed (Protein A capture) andhad a level of binding activity to human CD38.

TABLE 17 Anti-CD38- Variable Variable Protein A attenuated IFN HeavyLight K_(D) capture fusion protein SEQ ID NO: SEQ ID NO: (M) (RU) A5D1.0114 125 2.28 × 10⁻⁹ N/A (chimeric) A5D1.1 115 126 2.95 × 10⁻⁸ 175 A5D1.2115 127 2.95 × 10⁻⁸ 289 A5D1.3 115 128 2.35 × 10⁻⁸ 248 A5D1.4 115 1292.85 × 10⁻⁸ 427 A5D1.5 115 130 1.84 × 10⁻⁷ 269 A5D1.6 115 131 2.32 ×10⁻⁸ 338 A5D1.7 116 126 1.05 × 10⁻⁸ 132 A5D1.8 116 127 6.80 × 10⁻⁹ 263A5D1.9 116 128 9.93 × 10⁻⁸ 128 A5D1.10 116 129 5.69 × 10⁻⁹ 358 A5D1.11116 130 1.64 × 10⁻⁸ 250 A5D1.12 116 131 5.61 × 10⁻⁹ 345 A5D1.13 117 1261.44 × 10⁻⁸ 213 A5D1.14 117 127 1.52 × 10⁻⁸ 344 A5D1.15 117 128 1.46 ×10⁻⁸ 167 A5D1.16 117 129 1.37 × 10⁻⁸ 524 A5D1.17 117 130 3.28 × 10⁻⁸ 410A5D1.18 117 131 1.01 × 10⁻⁸ 396 A5D1.19 118 126 1.01 × 10⁻⁸ 245 A5D1.20118 127 1.07 × 10⁻⁸ 282 A5D1.21 118 128 7.94 × 10⁻⁹ 351 A5D1.22 118 1298.97 × 10⁻⁹ 566 A5D1.23 118 130 2.14 × 10⁻⁸ 336 A5D1.24 118 131 8.01 ×10⁻⁹ 319 A5D1.25 119 126 DNB 165 A5D1.26 119 127 DNB 286 A5D1.27 119 128DNB 265 A5D1.28 119 129 DNB 493 A5D1.29 119 130 DNB 275 A5D1.30 119 131DNB 263 A5D1.31 120 126 1.05 × 10⁻⁷ 206 A5D1.32 120 127 1.20 × 10⁻⁷ 318A5D1.33 120 128 9.83 × 10⁻⁸ 176 A5D1.34 120 129 1.06 × 10⁻⁷ 497 A5D1.35120 130 6.07 × 10⁻⁷ 211 A5D1.36 120 131 8.58 × 10⁻⁸ 331 A5D1.37 121 1261.01 × 10⁻⁷ 184 A5D1.38 121 127 1.21 × 10⁻⁷ 315 A5D1.39 121 128 9.55 ×10⁻⁸ 191 A5D1.40 121 129 1.22 × 10⁻⁷ 460 A5D1.41 121 130 5.60 × 10⁻⁷ 409A5D1.42 121 131 8.54 × 10⁻⁸ 301 A5D1.43 122 126 1.78 × 10⁻⁸ 150 A5D1.44122 127 1.76 × 10⁻⁸ 226 A5D1.45 122 128 1.42 × 10⁻⁸ 177 A5D1.46 122 1291.51 × 10⁻⁸ 401 A5D1.47 122 130 1.89 × 10⁻⁸ 364 A5D1.48 122 131 1.20 ×10⁻⁸ 273 A5D1.49 123 126 6.32 × 10⁻⁹ 141 A5D1.50 123 127 5.64 × 10⁻⁹ 212A5D1.51 123 128 4.97 × 10⁻⁹ 188 A5D1.52 123 129 4.07 × 10⁻⁹ 493 A5D1.53123 130 6.98 × 10⁻⁹ 561 A5D1.54 123 131 4.49 × 10⁻⁹ 253 A5D1.55 124 1266.48 × 10⁻⁹ 203 A5D1.56 124 127 8.44 × 10⁻⁹ 144 A5D1.57 124 128 5.59 ×10⁻⁹ 233 A5D1.58 124 129 5.37 × 10⁻⁹ 376 A5D1.59 124 130 1.05 × 10⁻⁸ 313A5D1.60 124 131 4.57 × 10⁻⁹ 429 DNB—did not bind. N/A—Not available

TABLE 18 Anti-CD38- Variable Variable Protein A attenuated IFN HeavyLight K_(D) capture fusion protein SEQ ID NO: SEQ ID NO: (M) (RU) A5E8.0(chimeric) 132 143 5.50 × 10⁻⁹ N/A A5E8.1 133 144 2.31 × 10⁻⁷ 267 A5E8.2133 145 2.37 × 10⁻⁷ 459 A5E8.3 133 146 3.59 × 10⁻⁷ 281 A5E8.4 133 147DNB 420 A5E8.5 134 144 1.75 × 10⁻⁷ 172 A5E8.6 134 145 1.57 × 10⁻⁷ 611A5E8.7 134 146 2.58 × 10⁻⁷ 201 A5E8.8 134 147 8.09 × 10⁻⁷ 308 A5E8.9 135144 1.05 × 10⁻⁸ 153 A5E8.10 135 145 2.13 × 10⁻⁸ 503 A5E8.11 135 146 2.69× 10⁻⁸ 372 A5E8.12 135 147 DNB 212 A5E8.13 136 144 3.98 × 10⁻⁸ 301A5E8.14 136 145 1.26 × 10⁻⁷ 543 A5E8.15 136 146 1.39 × 10⁻⁷ 504 A5E8.16136 147 DNB 284 A5E8.17 137 144 2.76 × 10⁻⁸ 397 A5E8.18 137 145 8.81 ×10⁻⁸ 430 A5E8.19 137 146 1.09 × 10⁻⁷ 220 A5E8.20 137 147 DNB 397 A5E8.21138 144 DNB 277 A5E8.22 138 145 DNB 409 A5E8.23 138 146 DNB 339 A5E8.24138 147 DNB 266 A5E8.25 139 144 DNB 283 A5E8.26 139 145 DNB 395 A5E8.27139 146 DNB 277 A5E8.28 139 147 DNB 290 A5E8.29 140 144 3.91 × 10⁻⁸ 207A5E8.30 140 145 5.00 × 10⁻⁸ 255 A5E8.31 140 146 6.61 × 10⁻⁸ 267 A5E8.32140 147 DNB 42 A5E8.33 141 144 1.12 × 10⁻⁸ 134 A5E8.34 141 145 1.63 ×10⁻⁸ 301 A5E8.35 141 146 1.85 × 10⁻⁸ 177 A5E8.36 141 147 DNB 10 A5E8.37142 144 8.19 × 10⁻⁸ 200 A5E8.38 142 145 1.55 × 10⁻⁸ 328 A5E8.39 142 1461.74 × 10⁻⁸ 232 A5E8.40 142 147 DNB 126 DNB—did not bind. N/A—notavailable

TABLE 19 Anti-CD38- SEQ ID: SEQ ID: Protein A attenuated IFN VariableVariable K_(D) capture fusion protein Heavy Light (M) (RU) A10A2.0 148157 5.98 × 10⁻¹⁰ N/A (chimeric) A10A2.1 149 158 DNB 728 A10A2.2 149 159DNB 689 A10A2.3 149 160 DNB 850 A10A2.4 149 161 DNB 996 A10A2.5 149 162DNB 761 A10A2.6 149 163 DNB 890 A10A2.7 149 164 DNB 725 A10A2.8 150 1587.18 × 10⁻⁷ 718 A10A2.9 150 159 6.62 × 10⁻⁷ 627 A10A2.10 150 160 9.13 ×10⁻⁷ 850 A10A2.11 150 161 2.37 × 10⁻⁷ 956 A10A2.12 150 162 1.18 × 10⁻⁶864 A10A2.13 150 163 6.80 × 10⁻⁷ 765 A10A2.14 150 164 DNB 645 A10A2.15151 158 1.15 × 10⁻⁷ 488 A10A2.16 151 159 8.11 × 10⁻⁸ 759 A10A2.17 151160 1.84 × 10⁻⁷ 684 A10A2.18 151 161 3.39 × 10^(−8/) 907 A10A2.19 151162 1.84 × 10⁻⁷ 831 A10A2.20 151 163 1.23 × 10⁻⁷ 560 A10A2.21 151 164DNB 337 A10A2.22 152 158 2.70 × 10⁻⁹ 890 A10A2.23 152 159 2.17 × 10⁻⁹828 A10A2.24 152 160 3.04 × 10⁻⁹ 803 A10A2.25 152 161 1.51 × 10⁻⁹ 1054A10A2.26 152 162 3.51 × 10⁻⁹ 741 A10A2.27 152 163 2.42 × 10⁻⁹ 603A10A2.28 152 164 3.69 × 10⁻⁸ 384 A10A2.29 153 158 2.77 × 10⁻⁸ 93A10A2.30 153 159 2.15 × 10⁻⁸ 86 A10A2.31 153 160 5.82 × 10⁻⁸ 33 A10A2.32153 161 8.49 × 10⁻⁹ 169 A10A2.33 153 162 5.66 × 10⁻⁸ 62 A10A2.34 153 1633.88 × 10⁻⁸ 56 A10A2.35 153 164 DNB DNE A10A2.36 154 158 8.38 × 10⁻⁹ 221A10A2.37 154 159 1.39 × 10⁻⁹ 858 A10A2.38 154 160 1.08 × 10⁻⁸ 178A10A2.39 154 161 3.80 × 10⁻⁹ 357 A10A2.40 154 162 1.34 × 10⁻⁸ 217A10A2.41 154 163 8.73 × 10⁻⁹ 202 A10A2.42 154 164 2.09 × 10⁻⁷ 175A10A2.43 154 158 2.45 × 10⁻⁷ 621 A10A2.44 155 159 6.23 × 10⁻⁹ 220A10A2.45 155 160 2.84 × 10⁻⁷ 881 A10A2.46 155 161 1.39 × 10⁻⁷ 1000A10A2.47 155 162 3.28 × 10⁻⁷ 9 A10A2.48 155 163 2.52 × 10⁻⁷ 565 A10A2.49155 164 DNB 499 A10A2.50 156 158 1.61 × 10⁻⁹ 567 A10A2.51 156 159 2.00 ×10⁻⁷ 603 A10A2.52 156 160 1.69 × 10⁻⁹ 723 A10A2.53 156 161 1.20 × 10⁻⁹729 A10A2.54 156 162 1.92 × 10⁻⁹ 639 A10A2.55 156 163 1.47 × 10⁻⁹ 692A10A2.56 156 164 1.97 × 10⁻⁷ 383 DNB—did not bind.DNE—did not express.

For each family of humanized antibodies—5D1, 5E8 and 10A2—severalhumanized heavy and light chain combinations failed to either expressprotein, or to bind to human CD38. A considerable number of antibodiesacross all 3 families of humanized antibodies expressed and bound tohuman CD38 with equilibrium dissociation constants in the nanomolar (nM)range. A10A2.53 and A10A2.25, which share a common light chain werechosen for further optimization. A10A2.53 was renamed A10.0 and A10A2.25was renamed A10.38.

Example 8 Improved Variants of A10.0

The A10.0 antibody was optimized through alterations to the variableheavy and/or light chain sequences with the aim of yielding a positiveeffect on the biophysical and in silico immunogenicity of the antibodywhilst causing minimal impact on the functional activity of theantibody.

In-Silico Immunogenicity Analysis of A10.0 Heavy- and Light Chains

In silico immunogenicity analyses of the A10.0 heavy- and light chainvariable regions were made using the Epibase software package. Severalamino acid substitutions were introduced into the heavy- and light chainvariable regions of A10.0 to remove potential immunogenic epitopes. Anamino acid sequence alignment of the heavy chain variable regionvariants produced aligned with the humanised heavy chain (SEQ ID NO:156) is shown in FIG. 9. An amino acid sequence alignment of the lightchain variable region variants aligned with the humanised light chain(SEQ ID NO: 161) is shown in FIG. 10. Details of the heavy- and lightchains variants co-expressed in HEK293E cells to produce proteins aresummarised in Table 20.

TABLE 20 VH Amino VK Anti- Acid Amino CD38- Sub- Variable Acid Variableattenuated stitution Heavy Sub- Light IFN (Relative SEQ stitution SEQfusion to ID (Relative to ID protein A10.0) NO: A10.0) NO: A10.1 A40E165 N/A 161 A10.2 A40G 166 N/A 161 A10.3 A40H 167 N/A 161 A10.4 A40Q 168N/A 161 A10.5 A40S 169 N/A 161 A10.6 A40V 170 N/A 161 A10.7 N35E 171 N/A161 A10.8 N35P 172 N/A 161 A10.9 N35Q 173 N/A 161 A10.10 N35S 174 N/A161 A10.11 R94E 175 N/A 161 A10.12 R94G 176 N/A 161 A10.13 R94P 177 N/A161 A10.14 R94T 178 N/A 161 A10.15 K96G 179 N/A 161 A10.16 K96T 180 N/A161 A10.17 N/A 156 K24E 181 A10.18 N/A 156 K24G 182 A10.19 N/A 156 K24P183 A10.20 N/A 156 K24Q 184 A10.21 N/A 156 R54D 185 A10.22 N/A 156 I48D186 A10.23 N/A 156 Y49E 187 A10.24 N/A 156 M89A 188 A10.25 N/A 156 M89E189 A10.26 N/A 156 M89H 190 A10.27 N/A 156 M89K 191 A10.28 N/A 156 M89P192 A10.29 N/A 156 M89Q 193 A10.30 N/A 156 M89S 194 A10.31 N/A 156 M89V195 A10.32 N/A 156 Q90D 196Each antibody generated using the heavy- and light chain pairingsoutlined in Table 20 was assessed for protein expression level andbinding to CD38 via SPR. Furthermore, potency assays were performedusing cell culture supernatants to assess the relative functionalactivity of each of these anti-CD38 antibody-attenuated IFN fusionproteins, Table 21.

TABLE 21 Annexin V (Fold Caspase change Assay CD38 relative (Fold CellAnti-CD38- binding by to change Pro- attenuated Protein A SPR (RU)untreated relative to liferation IFN fusion HPLC at 350 cells) untreatedAssay protein (mg/L) sec* Assay cells) (IC₅₀ pM) A10.1 16.9 1824 1.663.41 4078 A10.2 16.7 1821 1.66 5.19 7622 A10.3 25.0 2166 1.63 5.46 2148A10.4 23.7 2169 1.63 5.78 4108 A10.5 28.0 2240 1.64 5.80 3046 A10.6 31.02097 1.57 5.76 2283 A10.7 26.5 DNB 1.18 1.09 No IC₅₀ A10.8 2.4 DNB N/TN/T N/T A10.9 18.3 176 1.48 2.07 No IC₅₀ A10.10 32.2 1072 1.57 4.9718870 A10.11 28.3 98 1.57 3.64 No IC₅₀ A10.12 30.7 DNB 1.22 1.99 No IC₅₀A10.13 30.6 123 1.31 2.67 No IC₅₀ A10.14 30.5 247 1.19 5.11 68270 A10.1541.8 1254 1.52 5.44 5169 A10.16 24.2 1210 1.70 4.57 5224 A10.17 18.21686 1.79 6.11 3054 A10.18 32.5 2457 1.89 6.16 2178 A10.19 1.6 DNB 1.732.39 No IC₅₀ A10.20 12.2 1355 4.65 7.72 564 A10.21 19.9 1837 1.84 5.565330 A10.22 5.5 480 N/T N/T N/T A10.23 20.6 255 1.71 3.85 59720 A10.2434.6 1943 4.14 6.75 399 A10.25 28.3 1778 1.87 6.09 4910 A10.26 5.7 706N/T N/T N/T A10.27 7.4 136 N/T N/T N/T A10.28 2.2 48 N/T N/T N/T A10.2910.9 1443 N/T N/T No IC₅₀ A10.30 25.4 1865 1.98 6.21 1438 A10.31 5.8 469N/T N/T N/T A10.32 34.5 615 3.80 6.97 3628 The CD38 binding by SPRrefers to the amount of CD38 that remains bound to the surface after 350seconds of the dissociation phase. Annexin V Assay refer to cellspositively stained by Annexin V-FITC after 24 h treatment with antibodyconstructs at 20 nM. Caspase Assay refers to caspase activation of cellsafter 24 h treatment with antibody constructs at 20 nM. DNB—Did notbind; N/T—not tested; No IC₅₀-potency not sufficient for an IC₅₀ value.

Analyses of the amino acid sequences of the variable heavy- and lightchain sequences of A10.0 identified several potential deamidation sitesand one potential oxidation site. Variable heavy chain substitution N98Qwas prepared to remove a deamidation site from CDR3 of the heavy chain,SEQ ID NO: 197. A further variant of the A10.0 variable light chaincontaining the CDR2 substitution N53Q (SEQ ID NO: 198) was generated toremove this putative deamidation site. M89 within CDR3 of the lightchain was also altered through amino acid substitutions at this positionwith the combined aims of removing this potential oxidation site andreducing the predicted immunogenicity of this region of the light chain.These substitutions are outlined in Table 22, along with the heavy andlight chain pairings co-expressed to produce each anti-CD38-attenuatedIFN fusion protein.

TABLE 22 Amino Acid Anti-CD38- Substitution attenuated IFN (Relative toVariable Heavy Variable Light fusion protein A10.0) SEQ ID NO: SEQ IDNO: A10.35 Heavy Chain 197 161 N(98)Q A10.36 Light Chain 156 198 N(53)Q

Each antibody generated using the heavy- and light chain pairingsoutlined in Table 22 was assessed for protein expression level andbinding to CD38 via SPR. Furthermore, potency assays were performedusing cell culture supernatants to assess the relative functionalactivity of each of these anti-CD38 antibody-attenuated IFN fusionproteins, Table 23

TABLE 23 CD38 Annexin V Caspase binding assay (Fold Assay Anti-CD38-Protein by SPR change (Fold change Cell attenuated A (RU) relative torelative to Proliferation IFN fusion HPLC at 350 untreated untreatedAssay protein (mg/L) sec* cells)Assay cells) IC₅₀ (pM) A10.35 34.5 18891.83 6.16 6241 A10.36 52.4 1895 3.95 5.90 534.9 The CD38 binding by SPRrefers to the amount of CD38 that remains bound to the surface after 350seconds of the dissociation phase. Annexin V Assay refer to cellspositively stained by Annexin V-FITC after 24 h treatment with antibodyconstructs at 20 nM. Caspase Assay refers to caspase activation of cellsafter 24 h treatment with antibody constructs at 20 nM.

Example 9 Generating Improved Variants of A10.38

A10.0 and A10.38 share a common light chain. The optimized light chainsequences of A10.0 were paired with the heavy chain of the A10.38antibody with the aim of yielding a positive effect on the antibody'sbiophysical and in silico immunogenicity properties whilst having aminimal impact on functional activity. A summary of the changes and thepairings of heavy and light chains are described in Table 24.

TABLE 24 Anti-CD38- VK Amino Acid attenuated SEQ ID: Substitution SEQID: IFN fusion Variable (Relative to Variable protein Heavy A10.38)Light A10.38 152 N/A 161 A10.39 152 K24E 181 A10.40 152 K24G 182 A10.41152 K24P 183 A10.42 152 K24Q 184 A10.43 152 R54D 185 A10.44 152 I48D 186A10.45 152 Y49E 187 A10.46 152 M89A 188 A10.47 152 M89E 189 A10.48 152M89H 190 A10.49 152 M89K 191 A10.50 152 M89P 192 A10.51 152 M89Q 193A10.52 152 M89S 194 A10.53 152 M89V 195 A10.54 152 Q90D 196 A10.57 152N53Q 198

Each of the above antibodies was assessed for protein expression leveland binding to CD38 via SPR. Potency assays were performed using cellculture supernatants to assess the relative functional activity of eachof these anti-CD38 antibody-attenuated IFN fusion proteins, Table 25.

TABLE 25 Annexin V Caspase Assay (Fold Assay (Fold Anti-CD38- CD38change change attenuated Protein A binding by relative to relative toIFN fusion HPLC SPR (RU) untreated untreated protein (mg/L) at 350 sec*cells) cells) A10.38 119.7 1540 3.22 5.17 A10.39 113.4 1444 3.39 4.79A10.40 117.8 1562 3.28 5.12 A10.41 89.7 1459 3.27 5.12 A10.42 111.7 14433.32 5.60 A10.43 94.1 1426 3.21 6.15 A10.44 51.9 969 3.08 5.66 A10.45111.7 333 2.76 5.01 A10.46 120.0 1547 3.24 4.80 A10.47 107.3 1337 3.454.25 A10.48 45.5 865 3.06 5.48 A10.49 55.8 213 3.46 7.63 A10.50 11.3 1722.96 5.61 A10.51 51.6 1320 2.34 6.16 A10.52 70.0 1512 3.21 5.62 A10.5340.0 536 3.46 4.68 A10.54 61.3 583 3.10 6.20 A10.57 67.1 1431 3.06 6.04The CD38 binding by SPR refers to the amount of CD38 that remains boundto the surface after 350 seconds of the dissociation phase. Annexin VAssay refer to cells positively stained by Annexin V-FITC after 24 htreatment with antibody constructs at 20 nM. Caspase Assay refers tocaspase activation of cells after 24 h treatment with antibodyconstructs at 20 nM. N/T—Not Tested

Attenuated IFN is Required for Potent Apoptotic and Caspase Activationin Tumor Cell Lines

The relative potency of anti-CD38 antibodies A10.0 (attenuated IFNfusion) and X10.0 (no fusion) were compared using the Annexin V, Caspaseand the Cell Proliferation Assays outlined in Example 5. The relativepotency of A10.38 and X10.38 was also compared, Table 26.

TABLE 26 Annexin V Caspase Assay (Fold Assay (Fold Anti-CD38- changechange Cell attenuated relative to relative to Proliferation IFN fusionuntreated untreated Assay protein cells) cells) IC₅₀ (pM) Figures A10.02.10 4.23 2081 FIG. 18 X10.0 1.27 1.70 No IC50 FIG. 18 A10.38 3.22 5.171118 FIG. 25 X10.38 1.46 2.09 No IC50 FIG. 25 Annexin V Assay refer tocells positively stained by Annexin V-FITC after 24 h treatment withantibody constructs at 20 nM. Caspase Assay refers to caspase activationof cells after 24 h treatment with antibody constructs at 20 nM.

These data demonstrate the potent apoptotic activity exhibited byantibodies A10.0 and A10.38 relative to X10.0 and X10.38 respectivelynecessitates the presence of the attenuated IFN fusion. Noanti-proliferative activity was observed with antibodies without anattenuated IFN.

A consensus sequence alignment of heavy chain variable regions fromproteins with functional activity is shown in FIG. 11. A consensussequence alignment of light chain variable regions from proteins withfunctional activity is shown in FIG. 12. It could be further envisionedthat combinations of substitutions could be made such as those describedfor Anti-CD38 antibodies X10.60, X10.61, X10.62, X10.63, X10.64, X10.65,X10.66, X10.67, X10.68, X10.69, X10.70, X10.71, X10.72, X10.73, X10.74,X10.75, X10.76, X10.77, X10.78, X10.79, X10.80, X10.81, X10.82, X10.83,X10.84, X10.85, X10.86, X10.87, X10.88, X10.89, X10.90, X10.91, X10.92,X10.93, X10.94, X10.95, X10.96, X10.97, X10.98, X10.99, X10.100,X10.101, X10.102, X10.103, X10.104, X10.105, X10.106, X10.107, X10.108,X10.109, X10.110, X10.111, X10.112, X10.113, X10.114, X10.115, X10.116,X10.117, X10.118, X10.119, X10.120, X10.121, X10.122, X10.123, X10.124,X10.125, X10.126, X10.127, X10.128, X10.129, X10.130, X10.131, X10.132,X10.133, X10.134, X10.135, X10.136, X10.137, X10.138, X10.139, X10.140,X10.141, X10.142, X10.143, X10.144, X10.145, X10.146, X10.147 (FIG. 11,FIG. 12). Further the above Anti-CD38 antibodies could also beconstructed as Anti-CD38-attenuated IFN fusion proteins and tested forfunctional activity as described herein.

H929 Multiple Myeloma Xenograft Model

The in vivo potency of 10A2 variants A10.0 and A10A2.0 were evaluated inan NCI-H929 s.c. mouse multiple myeloma model, FIG. 27. Both were shownto have potent anti-tumour activity in this model. Such a model could beused to test for anti-tumor activity of other protein constructsdescribed within.

Off-Target Activity for the 10A2 Variants

The off-target activity of the 10A2 variants A10.0, A10.38, A10A2.37 andA10A2.39 in comparison with the parental A10A2.0 chimeric antibody fusedto wildtype and attenuated interferon 145D was assessed in either theiLite reporter gene assay and/or the HEK Blue assay and is shown in FIG.28 and FIG. 29. The EC₅₀ values are provided in FIG. 28 and FIG. 29. Theoff-target activity confirms the attenuation of the interferon and theneed for the antibody to be targeted to CD38 to restore function.

Further In-Vitro Potency Data for A10.0 and Related Constructs

A selection of the above Anti-CD38-attenuated IFN fusion proteins werepurified and analysed for binding to CD38 positive cells in cell basedassays. In addition potency assays were repeated to more accuratelydetermine the relative activity of each of these Anti-CD38-attenuatedIFN fusion proteins. The methods for these various assays are describedin Example 5. The results of each of these assays are given in Table 27.

TABLE 27 Annexin V Caspase Assay (Fold Assay (Fold Anti-CD38- H929 Flowchange change Cell attenuated binding relative to relative toProliferation IFN fusion (EC₅₀ in untreated untreated Assay IC₅₀ proteinμg/mL) cells) cells) (pM) Figures A10.0 1.49 3.05 1.38 120.8 17, 18, 21,22, 23, 24, 25, 26, 27 28, 29 A10.1 1.03 1.34 4.63 63.6 24, 25 A10.20.57 1.37 4.37 45.6 24, 25 A10.3 0.55 1.43 5.52 65.8 24, 25 A10.5 0.483.27 1.61 53.79 24, 25 A10.6 0.35 3.16 1.66 98.85 24, 25 A10.10 6.843.02 1.64 1967.00 24, 25 A10.14 2.26 2.46 1.56 2207 22, 24, 25 A10.152.06 3.06 2.55 174.4 22, 24, 25 A10.16 1.17 1.35 5.18 49.5 24, 25 A10.181.03 2.95 1.70 124.2 22, 24, 25 A10.20 2.11 2.84 1.26 656 24, 25 A10.210.78 3.0 1.39 147.3 22, 24, 25 A10.24 0.81 2.95 7.47 87.99 22, 24, 25A10.25 1.37 2.75 1.38 27.69 24, 25 A10.30 0.88 3.22 1.60 18.73 24, 25A10.32 51.69 1.93 1.19 381.4 24, 25 A10.35 1.10 2.97 2.05 93.96 22, 24,25 A10.36 1.53 3.21 3.61 57.83 22, 24, 25 A10.37 18.57 2.53 1.40 163.524, 25 A10.38 1.13 3.27 1.53 36.79 23, 24, 25, 26, 29 A10.40 0.99 1.445.16 3.02 24, 25 A10.42 1.61 1.58 1.78 155.3 24, 25 A10.43 1.20 1.641.79 120.9 24, 25 A10.44 1.65 1.58 1.91 308.6 24, 25 A10.46 0.73 1.845.99 2.63 24, 25 A10.47 0.79 1.70 1.53 5.707 24, 25 A10.48 1.72 1.581.60 22.33 24, 25 A10.53 1.32 1.56 1.67 56.01 24, 25 A10.54 5.90 1.521.43 2008 24, 25 A10.56 2.43 1.84 1.95 141.7 24, 25 A10.57 1.54 1.494.71 126.1 24, 25 A10.59 0.89 2.48 2.38 45.75 21, 24, 25 The flowbinding was determined in H929 cell line. Annexin V Assay refer to cellspositively stained by Annexin V-FITC after 24 h treatment with antibodyconstructs at 20 nM. Caspase Assay refers to caspase activation of cellsafter 24 h treatment with antibody constructs at 20 nM.

Anti-CD38-Attenuated IFN Fusion Protein with Alternative Constant Region

A10.0 comprises an anti-CD38-attenuated IFN fusion protein in which theconstant region of the protein is HC-L0-IFN-alpha (A145D) IgG4 (SEQ IDNO: 9). Using gene synthesis, the constant region of this protein wasreplaced with HC-L0-IFN-alpha (A145D) IgG1 (SEQ ID NO: 10), paired withA10.0 light chain (SEQ ID NO: 161) and given the designation A10.59. Theprotein was expressed and was found to be potent in functional assays(Table 28). While the majority of the proteins tested in the foregoingexamples were constructed on the human IgG4 constant region, these datademonstrate that other antibody constant regions, such as human IgG1,may also be used, with the resultant antibody-attenuated IFN fusionconstruct having potent biologic activity equivalent to constructs thatutilize a human IgG4 constant region.

TABLE 28 Annexin V Caspase Anti- H929 Assay (Fold Assay(Fold CD38- Flowchange change Cell attenuated binding relative to relative toProliferation IFN fusion (EC₅₀in untreated untreated Assay proteinμg/mL) cells) cells) IC₅₀ (pM)* Figures A10.0 1.50 3.05 1.89 2081 21,24, 25 A10.59 0.89 2.48 2.38 328.6 21, 24, 25 Annexin V Assay refer tocells positively stained by Annexin V-FITC after 24 h treatment withantibody constructs at 20 nM. Caspase Assay refers to caspase activationof cells after 24 h treatment with antibody constructs at 20 nM. N/T isNot Tested, *Data obtained from Cell Proliferation Assay assessed withcell culture supernatant.

Table 29 lists the pairing of variable heavy chain, variable light chainand constant region for each antibody described herein.

TABLE 29 Variable Heavy Chain Variable Light SEQ Constant Heavy SEQ IDNO: Region SEQ ID NO: (amino ID NO: Antibody Name (amino acid) acid)(amino acid) A02.10 19 14 9 A02.11 20 14 9 A02.112 34 65 10 A02.12 34 659 A02.13 35 65 9 A02.16 34 92 9 A02.17 34 93 9 A02.18 34 73 9 A02.19 3474 9 A02.2 13 65 9 A02.20 34 75 9 A02.21 34 76 9 A02.22 34 77 9 A02.2334 78 9 A02.24 34 79 9 A02.25 34 80 9 A02.26 34 81 9 A02.27 34 82 9A02.28 34 83 9 A02.29 34 84 9 A02.3 17 65 9 A02.30 34 85 9 A02.31 34 869 A02.32 34 87 9 A02.33 34 88 9 A02.34 34 89 9 A02.35 34 90 9 A02.36 3491 9 A02.37 34 66 9 A02.38 34 113 9 A02.39 34 112 9 A02.4 18 65 9 A02.40111 65 9 A02.41 110 65 9 A02.43 110 113 9 A02.44 111 112 9 A02.46 34 679 A02.47 34 68 9 A02.48 34 69 9 A02.49 34 70 9 A02.5 19 65 9 A02.50 3471 9 A02.51 34 72 9 A02.52 34 94 9 A02.53 34 95 9 A02.54 34 96 9 A02.5534 97 9 A02.56 34 98 9 A02.57 34 99 9 A02.58 34 100 9 A02.59 34 101 9A02.6 20 65 9 A02.60 34 102 9 A02.61 34 103 9 A02.62 34 104 9 A02.63 34105 9 A02.64 34 106 9 A02.65 34 107 9 A02.66 34 108 9 A02.67 34 109 9A02.8 17 14 9 A02.9 18 14 9 A10.1 165 161 9 A10.10 174 161 9 A10.11 175161 9 A10.12 176 161 9 A10.13 177 161 9 A10.14 178 161 9 A10.15 179 1619 A10.16 180 161 9 A10.17 156 181 9 A10.18 156 182 9 A10.19 156 183 9A10.2 166 161 9 A10.20 156 184 9 A10.21 156 185 9 A10.22 156 186 9A10.23 156 187 9 A10.24 156 188 9 A10.25 156 189 9 A10.26 156 190 9A10.27 156 191 9 A10.28 156 192 9 A10.29 156 193 9 A10.3 167 161 9A10.30 156 194 9 A10.31 156 195 9 A10.32 156 196 9 A10.35 197 161 9A10.36 156 198 9 A10.38 152 161 9 A10.39 152 181 9 A10.4 168 161 9A10.40 152 182 9 A10.41 152 183 9 A10.42 152 184 9 A10.43 152 185 9A10.44 152 186 9 A10.45 152 187 9 A10.46 152 188 9 A10.47 152 189 9A10.48 152 190 9 A10.49 152 191 9 A10.5 169 161 9 A10.50 152 192 9A10.51 152 193 9 A10.52 152 194 9 A10.53 152 195 9 A10.54 152 196 9A10.57 152 198 9 A10.59 156 161 10 A10.6 170 161 9 A10.7 171 161 9 A10.8172 161 9 A10.9 173 161 9 A10A2.0 (chimeric) 148 157 9 A10A2.1 149 158 9A10A2.10 150 160 9 A10A2.11 150 161 9 A10A2.12 150 162 9 A10A2.13 150163 9 A10A2.14 150 164 9 A10A2.15 151 158 9 A10A2.16 151 159 9 A10A2.17151 160 9 A10A2.18 151 161 9 A10A2.19 151 162 9 A10A2.2 149 159 9A10A2.20 151 163 9 A10A2.21 151 164 9 A10A2.22 152 158 9 A10A2.23 152159 9 A10A2.24 152 160 9 A10A2.25 152 161 9 A10A2.26 152 162 9 A10A2.27152 163 9 A10A2.28 152 164 9 A10A2.29 153 158 9 A10A2.3 149 160 9A10A2.30 153 159 9 A10A2.31 153 160 9 A10A2.32 153 161 9 A10A2.33 153162 9 A10A2.34 153 163 9 A10A2.35 153 164 9 A10A2.36 154 158 9 A10A2.37154 159 9 A10A2.38 154 160 9 A10A2.39 154 161 9 A10A2.4 149 161 9A10A2.40 154 162 9 A10A2.41 154 163 9 A10A2.42 154 164 9 A10A2.43 154158 9 A10A2.44 155 159 9 A10A2.45 155 160 9 A10A2.46 155 161 9 A10A2.47155 162 9 A10A2.48 155 163 9 A10A2.49 155 164 9 A10A2.5 149 162 9A10A2.50 156 158 9 A10A2.51 156 159 9 A10A2.52 156 160 9 A10A2.53 156161 9 A10A2.54 156 162 9 A10A2.55 156 163 9 A10A2.56 156 164 9 A10A2.6149 163 9 A10A2.7 149 164 9 A10A2.8 150 158 9 A10A2.9 150 159 9 A5D1.0(chimeric) 114 125 9 A5D1.1 115 126 9 A5D1.10 116 129 9 A5D1.11 116 1309 A5D1.12 116 131 9 A5D1.13 117 126 9 A5D1.14 117 127 9 A5D1.15 117 1289 A5D1.16 117 129 9 A5D1.17 117 130 9 A5D1.18 117 131 9 A5D1.19 118 1269 A5D1.2 115 127 9 A5D1.20 118 127 9 A5D1.21 118 128 9 A5D1.22 118 129 9A5D1.23 118 130 9 A5D1.24 118 131 9 A5D1.25 119 126 9 A5D1.26 119 127 9A5D1.27 119 128 9 A5D1.28 119 129 9 A5D1.29 119 130 9 A5D1.3 115 128 9A5D1.30 119 131 9 A5D1.31 120 126 9 A5D1.32 120 127 9 A5D1.33 120 128 9A5D1.34 120 129 9 A5D1.35 120 130 9 A5D1.36 120 131 9 A5D1.37 121 126 9A5D1.38 121 127 9 A5D1.39 121 128 9 A5D1.4 115 129 9 A5D1.40 121 129 9A5D1.41 121 130 9 A5D1.42 121 131 9 A5D1.43 122 126 9 A5D1.44 122 127 9A5D1.45 122 128 9 A5D1.46 122 129 9 A5D1.47 122 130 9 A5D1.48 122 131 9A5D1.49 123 126 9 A5D1.5 115 130 9 A5D1.50 123 127 9 A5D1.51 123 128 9A5D1.52 123 129 9 A5D1.53 123 130 9 A5D1.54 123 131 9 A5D1.55 124 126 9A5D1.56 124 127 9 A5D1.57 124 128 9 A5D1.58 124 129 9 A5D1.59 124 130 9A5D1.6 115 131 9 A5D1.60 124 131 9 A5D1.7 116 126 9 A5D1.8 116 127 9A5D1.9 116 128 9 A5E8.0 (chimeric) 132 143 9 A5E8.1 133 144 9 A5E8.10135 145 9 A5E8.11 135 146 9 A5E8.12 135 147 9 A5E8.13 136 144 9 A5E8.14136 145 9 A5E8.15 136 146 9 A5E8.16 136 147 9 A5E8.17 137 144 9 A5E8.18137 145 9 A5E8.19 137 146 9 A5E8.2 133 145 9 A5E8.20 137 147 9 A5E8.21138 144 9 A5E8.22 138 145 9 A5E8.23 138 146 9 A5E8.24 138 147 9 A5E8.25139 144 9 A5E8.26 139 145 9 A5E8.27 139 146 9 A5E8.28 139 147 9 A5E8.29140 144 9 A5E8.3 133 146 9 A5E8.30 140 145 9 A5E8.31 140 146 9 A5E8.32140 147 9 A5E8.33 141 144 9 A5E8.34 141 145 9 A5E8.35 141 146 9 A5E8.36141 147 9 A5E8.37 142 144 9 A5E8.38 142 145 9 A5E8.39 142 146 9 A5E8.4133 147 9 A5E8.40 142 147 9 A5E8.5 134 144 9 A5E8.6 134 145 9 A5E8.7 134146 9 A5E8.8 134 147 9 A5E8.9 135 144 9 X02.10 19 14 3 X02.100 13 58 3X02.101 13 59 3 X02.102 13 60 3 X02.103 13 61 3 X02.104 13 62 3 X02.10513 63 3 X02.106 13 64 3 X02.107 13 65 3 X02.108 32 14 3 X02.11 20 14 3X02.110 33 14 3 X02.114 13 660 3 X02.115 13 661 3 X02.116 13 662 3X02.117 13 663 3 X02.118 34 700 3 X02.119 34 701 3 X02.120 728 700 3X02.121 729 700 3 X02.122 730 700 3 X02.123 731 700 3 X02.124 728 701 3X02.125 729 701 3 X02.126 730 701 3 X02.127 731 701 3 X02.68 21 14 3X02.69 22 14 3 X02.70 23 14 3 X02.71 24 14 3 X02.72 25 14 3 X02.73 26 143 X02.74 27 14 3 X02.75 28 14 3 X02.76 29 14 3 X02.77 30 14 3 X02.78 3114 3 X02.8 17 14 3 X02.80 13 38 3 X02.81 13 39 3 X02.82 13 40 3 X02.8313 41 3 X02.84 13 42 3 X02.85 13 43 3 X02.86 13 44 3 X02.87 13 45 3X02.88 13 46 3 X02.89 13 47 3 X02.9 18 14 3 X02.90 13 48 3 X02.91 13 493 X02.92 13 50 3 X02.93 13 51 3 X02.94 13 52 3 X02.95 13 53 3 X02.96 1354 3 X02.97 13 55 3 X02.98 13 56 3 X02.99 13 57 3 X10.100 720 706 3X10.101 721 706 3 X10.102 722 706 3 X10.103 723 706 3 X10.104 739 706 3X10.105 740 706 3 X10.106 741 706 3 X10.107 742 706 3 X10.108 720 707 3X10.109 721 707 3 X10.110 722 707 3 X10.111 723 707 3 X10.112 739 707 3X10.113 740 707 3 X10.114 741 707 3 X10.115 742 707 3 X10.116 720 708 3X10.117 721 708 3 X10.118 722 708 3 X10.119 723 708 3 X10.120 739 708 3X10.121 740 708 3 X10.122 741 708 3 X10.123 742 708 3 X10.124 720 709 3X10.125 721 709 3 X10.126 722 709 3 X10.127 723 709 3 X10.128 739 709 3X10.129 740 709 3 X10.130 741 709 3 X10.131 742 709 3 X10.132 720 710 3X10.133 721 710 3 X10.134 722 710 3 X10.135 723 710 3 X10.136 739 710 3X10.137 740 710 3 X10.138 741 710 3 X10.139 742 710 3 X10.140 720 711 3X10.141 721 711 3 X10.142 722 711 3 X10.143 723 711 3 X10.144 739 711 3X10.145 740 711 3 X10.146 741 711 3 X10.147 742 711 3 X10.60 156 704 3X10.61 156 705 3 X10.62 156 706 3 X10.63 156 707 3 X10.64 156 708 3X10.65 156 709 3 X10.66 156 710 3 X10.67 156 711 3 X10.68 720 161 3X10.69 721 161 3 X10.70 722 161 3 X10.71 723 161 3 X10.72 739 161 3X10.73 740 161 3 X10.74 741 161 3 X10.75 742 161 3 X10.76 152 704 3X10.77 152 705 3 X10.78 152 706 3 X10.79 152 707 3 X10.80 152 708 3X10.81 152 709 3 X10.82 152 710 3 X10.83 152 711 3 X10.84 720 704 3X10.85 721 704 3 X10.86 722 704 3 X10.87 723 704 3 X10.88 739 704 3X10.89 740 704 3 X10.90 741 704 3 X10.91 742 704 3 X10.92 720 705 3X10.93 721 705 3 X10.94 722 705 3 X10.95 723 705 3 X10.96 739 705 3X10.97 740 705 3 X10.98 741 705 3 X10.99 742 705 3 X910/12-HC-L0-IFN-110 112 9 alpha (A145D) IgG4 X913/15-HC-L0-IFN- 111 113 9 alpha (A145D)IgG4

TABLE 30 SEQ ID NO: Type Description 1 AA Human CD38 2 AA CynomolgusCD38 3 AA Human IgG4 constant heavy chain 4 AA Human IgG1 constant heavychain 5 AA Human kappa constant region 6 AA Human lambda constant region7 AA IFN-alpha2b 8 AA Intron A 9 AA HC-L0-IFN-alpha (A145D) IgG4 10 AAHC-L0-IFN-alpha (A145D) IgG1 11 AA A02.1 heavy chain 12 AA A02.1 lightchain 13 AA A02.1 variable heavy chain 14 AA A02.1 variable light chain15 AA X02.1VH variable heavy chain 16 AA IGHV4-61*01 germline sequence17 AA X02.8VH variable heavy chain 18 AA X02.9VH variable heavy chain 19AA X02.10VH variable heavy chain 20 AA X02.11VH variable heavy chain 21AA X02.68VH variable heavy chain 22 AA X02.69VH variable heavy chain 23AA X02.70VH variable heavy chain 24 AA X02.71VH variable heavy chain 25AA X02.72VH variable heavy chain 26 AA X02.73VH variable heavy chain 27AA X02.74VH variable heavy chain 28 AA X02.75VH variable heavy chain 29AA X02.76VH variable heavy chain 30 AA X02.77VH variable heavy chain 31AA X02.78VH variable heavy chain 32 AA X02.108VH variable heavy chain 33AA X02.110VH variable heavy chain 34 AA A02.12VH variable heavy chain 35AA A02.13VH variable heavy chain 36 AA A02.1VL variable light chain 37AA IGLV5-37*01 germline sequence 38 AA X02.80VL variable light chain 39AA X02.81VL variable light chain 40 AA X02.82VL variable light chain 41AA X02.83VL variable light chain 42 AA X02.84VL variable light chain 43AA X02.85VL variable light chain 44 AA X02.86VL variable light chain 45AA X02.87VL variable light chain 46 AA X02.88VL variable light chain 47AA X02.89VL variable light chain 48 AA X02.90VL variable light chain 49AA X02.91VL variable light chain 50 AA X02.92VL variable light chain 51AA X02.93VL variable light chain 52 AA X02.94VL variable light chain 53AA X02.95VL variable light chain 54 AA X02.96VL variable light chain 55AA X02.97VL variable light chain 56 AA X02.98VL variable light chain 57AA X02.99VL variable light chain 58 AA X02.100VL variable light chain 59AA X02.101VL variable light chain 60 AA X02.102VL variable light chain61 AA X02.103VL variable light chain 62 AA X02.104VL variable lightchain 63 AA X02.105VL variable light chain 64 AA X02.106VL variablelight chain 65 AA X02.107VL variable light chain 66 AA A02.37VL variablelight chain 67 AA A02.46VL variable light chain 68 AA A02.47VL variablelight chain 69 AA A02.48VL variable light chain 70 AA A02.49VL variablelight chain 71 AA A02.50VL variable light chain 72 AA A02.51VL variablelight chain 73 AA A02.18VL variable light chain 74 AA A02.19VL variablelight chain 75 AA A02.20VL variable light chain 76 AA A02.21VL variablelight chain 77 AA A02.22VL variable light chain 78 AA A02.23VL variablelight chain 79 AA A02.24VL variable light chain 80 AA A02.25VL variablelight chain 81 AA A02.26VL variable light chain 82 AA A02.27VL variablelight chain 83 AA A02.28VL variable light chain 84 AA A02.29VL variablelight chain 85 AA A02.30VL variable light chain 86 AA A02.31VL variablelight chain 87 AA A02.32VL variable light chain 88 AA A02.33VL variablelight chain 89 AA A02.34VL variable light chain 90 AA A02.35VL variablelight chain 91 AA A02.36VL variable light chain 92 AA X02.16VL variablelight chain 93 AA X02.17VL variable light chain 94 AA A02.52VL variablelight chain 95 AA A02.53VL variable light chain 96 AA A02.54VL variablelight chain 97 AA A02.55VL variable light chain 98 AA A02.56VL variablelight chain 99 AA A02.57VL variable light chain 100 AA A02.58VL variablelight chain 101 AA A02.59VL variable light chain 102 AA A02.60VLvariable light chain 103 AA A02.61VL variable light chain 104 AAA02.62VL variable light chain 105 AA A02.63VL variable light chain 106AA A02.64VL variable light chain 107 AA A02.65VL variable light chain108 AA A02.66VL variable light chain 109 AA A02.67VL variable lightchain 110 AA 910VH variable heavy chain 111 AA 915 VH variable heavychain 112 AA 912VL variable light chain 113 AA 913VL variable lightchain 114 AA Chimeric 5D1-E2-VH 115 AA 5d1_1-f*01VH 116 AA5d1_1-f*01VH94R 117 AA 5d1_1-18*01VH 118 AA 5d1_1-18*01VH71A 119 AA5d1_1-24*01VH 120 AA 5d1_1-24*01VH71A 121 AA 5d1_1-24*01VH29F 122 AA5d1_1-24*01VH94R 123 AA 5d1_1-45*01VH 124 AA 5d1_1-45*01VH71A 125 AAChimeric 5D1VK 126 AA 5d1_1-5*01VK 127 AA 5d1_1-9*01VK 128 AA5d1_1-12*01VK 129 AA 5d1_1D-13*01VK 130 AA 5d1_1D-16*01VK 131 AA5d1_3-15*01VK 132 AA Chimeric 5E8 133 AA 5E8-1-f*01VH 134 AA5E8-1-f*01VH30I 135 AA 5E8-1-f*01VH94R 136 AA 5E8-1-18*01VH 137 AA5E8-1-18*01VH71A 138 AA 5E8-1-24*01VH 139 AA 5E8-1-24*01VH71A 140 AA5E8-1-24*01VH94R 141 AA 5E8-1-45*01VH 142 AA 5E8-1-45*01VH71A 143 AAchimeric 5E8VK 144 AA 5E8-2-24*01VK 145 AA 5E8-2D-28*01VK 146 AA5E8-2D-29*01VK 147 AA 5E8-2-30*01VK 148 AA 10A2 chimeric VH 149 AA10A2_1-24*01VH 150 AA 10A2_1-24*01VH71A 151 AA 10A2_1-24*01VH94R 152 AA10A2_1-24*0171A94R 153 AA 10A2_1-45*01VH 154 AA 10A2_1-45*01VH71A 155 AA10A2_1-f*01VH 156 AA 10A2_1-f*01VH94R 157 AA 10A2 chimeric VK 158 AA10A2_1-9*01Vk 159 AA 10A2_1-12*01Vk 160 AA 10A2_1D-13*01Vk 161 AA10A2_1-33*01Vk 162 AA 10A2_3-11*02Vk 163 AA 10A2_3-15*01Vk 164 AA10A2_6-21*01Vk 165 AA 10A2VH + A40E 166 AA 10A2VH + A40G 167 AA 10A2VH +A40H 168 AA 10A2VH + A40Q 169 AA 10A2VH + A40S 170 AA 10A2VH + A40V 171AA 10A2VH + N35E 172 AA 10A2VH + N35P 173 AA 10A2VH + N35Q 174 AA10A2VH + N35S 175 AA 10A2VH + R94E 176 AA 10A2VH + R94G 177 AA 10A2VH +R94P 178 AA 10A2VH + R94T 179 AA 10A2VH + K96G 180 AA 10A2VH + K96T 181AA 10A2VK + K24E 182 AA 10A2VK + K24G 183 AA 10A2VK + K24P 184 AA10A2VK + K24Q 185 AA 10A2VK + R54D 186 AA 10A2VK + I48D 187 AA 10A2VK +Y49E 188 AA 10A2VK + M89A 189 AA 10A2VK + M89E 190 AA 10A2VK + M89H 191AA 10A2VK + M89K 192 AA 10A2VK + M89P 193 AA 10A2VK + M89Q 194 AA10A2VK + M89S 195 AA 10A2VK + M89V 196 AA 10A2VK + Q90D 197 AA 10A2VH(AQ) + N98Q 198 AA 10A2VK (AV) + N53Q 199 AA X02.1VH FWR1 200 AA X02.1VHCDR1 201 AA X02.1VH FWR2 202 AA X02.1VH CDR2 203 AA X02.1VH FWR3 204 AAX02.1VH CDR3 205 AA X02.1VH FWR4 206 AA IGHV4-61*01 FWR1 207 AAIGHV4-61*01 CDR1 208 AA IGHV4-61*01 FWR2 209 AA IGHV4-61*01 FWR3 210 AAX02.8VH FWR3 211 AA X02.9VH FWR2 212 AA X02.10VH FWR3 213 AA X02.11VHFWR3 214 AA X02.68VH FWR1 215 AA X02.69VH FWR1 216 AA X02.70VH FWR3 217AA X02.71VH FWR1 218 AA X02.72VH FWR3 219 AA X02.73VH FWR1 220 AAX02.74VH CDR3 221 AA X02.75VH FWR3 222 AA X02.76VH CDR3 223 AA X02.77VHCDR3 224 AA X02.78VH CDR1 225 AA X02.108 FWR3 226 AA X02.110VH FWR3 227AA A02.12VH FWR3 228 AA A02.12VH CDR3 229 AA A02.13VH FWR2 230 AAA02.13VH FWR3 231 AA A02.13VH CDR3 232 AA A02.1VL FWR1 233 AA A02.1VLCDR1 234 AA A02.1VL FWR2 235 AA A02.1VL CDR2 236 AA A02.1VL FWR3 237 AAA02.1VL CDR3 238 AA A02.1VL FWR4 239 AA IGLV5-37*01 FWR1 240 AAIGLV5-37*01 CDR1 241 AA IGLV5-37*01 CDR2 242 AA IGLV5-37*01 FWR3 243 AAIGLV5-37*01 CDR3 244 AA X02.80VL CDR3 245 AA X02.81VL FWR3 246 AAX02.82VL FWR2 247 AA X02.83VL FWR1 248 AA X02.84VL FWR2 249 AA X02.84VLCDR2 250 AA X02.86VL CDR1 251 AA X02.87VL CDR3 252 AA X02.88VL CDR1 253AA X02.89VL CDR2 254 AA X02.90VL CDR3 255 AA X02.91VL CDR1 256 AAX02.92VL CDR3 257 AA X02.93VL CDR3 258 AA X02.94VL CDR3 259 AA X02.95VLFWR1 260 AA X02.96VL FWR1 261 AA X02.97VL FWR1 262 AA X02.98VL CDR1 263AA X02.99VL CDR1 264 AA X02.100VL CDR2 265 AA X02.101VL FWR3 266 AAX02.102VL FWR3 267 AA X02.103VL FWR3 268 AA X02.104VL CDR3 269 AAX02.105VL CDR3 270 AA X02.106VL CDR3 271 AA X02.107VL FWR3 272 AAA02.37VL FWR3 273 AA A02.37VL CDR3 274 AA A02.46VL FWR3 275 AA A02.46VLCDR3 276 AA A02.47VL FWR3 277 AA A02.48VL FWR3 278 AA A02.49VL FWR3 279AA A02.50VL FWR3 280 AA A02.51VL FWR3 281 AA A02.18VL FWR2 282 AAA02.18VL FWR3 283 AA A02.19VL FWR2 284 AA A02.19VL FWR3 285 AA A02.20VLFWR2 286 AA A02.20VL FWR3 287 AA A02.21VL FWR2 288 AA A02.21VL FWR3 289AA A02.22VL FWR2 290 AA A02.22VL FWR3 291 AA A02.23VL FWR2 292 AAA02.23VL FWR3 293 AA A02.24VL FWR2 294 AA A02.24VL FWR3 295 AA A02.25VLFWR2 296 AA A02.25VL FWR3 297 AA A02.26VL FWR2 298 AA A02.26VL FWR3 299AA A02.27VL CDR2 300 AA A02.27VL FWR3 301 AA A02.28VL CDR2 302 AAA02.28VL FWR3 303 AA A02.29VL CDR2 304 AA A02.29VL FWR3 305 AA A02.30VLCDR2 306 AA A02.30VL FWR3 307 AA A02.31VL CDR2 308 AA A02.31VL FWR3 309AA A02.32VL CDR2 310 AA A02.32VL FWR3 311 AA A02.33VL CDR2 312 AAA02.33VL FWR3 313 AA A02.34VL CDR2 314 AA A02.34VL FWR3 315 AA A02.35VLCDR2 316 AA A02.35VL FWR3 317 AA A02.36VL CDR2 318 AA A02.36VL FWR3 319AA X02.16VL CDR1 320 AA X02.16VL FWR3 321 AA X02.16VL CDR3 322 AAX02.17VL CDR1 323 AA X02.17VL FWR3 324 AA X02.17VL CDR3 325 AA A02.52VLCDR1 326 AA A02.52VL CDR2 327 AA A02.52VL FWR3 328 AA A02.52VL CDR3 329AA A02.53VL CDR1 330 AA A02.53VL CDR2 331 AA A02.53VL FWR3 332 AAA02.53VL CDR3 333 AA A02.54VL CDR1 334 AA A02.54VL CDR2 335 AA A02.54VLFWR3 336 AA A02.54VL CDR3 337 AA A02.55VL CDR1 338 AA A02.55VL CDR2 339AA A02.55VL FWR3 340 AA A02.55VL CDR3 341 AA A02.56VL CDR1 342 AAA02.56VL CDR2 343 AA A02.56VL FWR3 344 AA A02.56VL CDR3 345 AA A02.57VLCDR1 346 AA A02.57VL CDR2 347 AA A02.57VL FWR3 348 AA A02.57VL CDR3 349AA A02.58VL CDR1 350 AA A02.58VL CDR2 351 AA A02.58VL FWR3 352 AAA02.58VL CDR3 353 AA A02.59VL CDR1 354 AA A02.59VL CDR2 355 AA A02.59VLFWR3 356 AA A02.59VL CDR3 357 AA A02.60VL CDR1 358 AA A02.60VL CDR2 359AA A02.60VL FWR3 360 AA A02.60VL CDR3 361 AA A02.61VL CDR1 362 AAA02.61VL CDR2 363 AA A02.61VL FWR3 364 AA A02.61VL CDR3 365 AA A02.62VLCDR1 366 AA A02.62VL CDR2 367 AA A02.62VL FWR3 368 AA A02.62VL CDR3 369AA A02.63VL CDR1 370 AA A02.63VL CDR2 371 AA A02.63VL FWR3 372 AAA02.63VL CDR3 373 AA A02.64VL CDR1 374 AA A02.64VL CDR2 375 AA A02.64VLFWR3 376 AA A02.64VL CDR3 377 AA A02.65VL CDR1 378 AA A02.65VL CDR2 379AA A02.65VL FWR3 380 AA A02.65VL CDR3 381 AA A02.66VL CDR1 382 AAA02.66VL CDR2 383 AA A02.66VL FWR3 384 AA A02.66VL CDR3 385 AA A02.67VLCDR1 386 AA A02.67VL CDR2 387 AA A02.67VL FWR3 388 AA A02.67VL CDR3 389AA 5D1.1VH FWR1 390 AA 5D1.1VH CDR1 391 AA 5D1.1VH FWR2 392 AA 5D1.1VHCDR2 393 AA 5D1.1VH FWR3 394 AA 5D1.1VH CDR3 395 AA 5D1.1VH FWR4 396 AA5D1.2VH FWR1 397 AA 5D1.2VH FWR2 398 AA 5D1.2VH CDR2 399 AA 5D1.2VH FWR3400 AA 5D1.3VH FWR1 401 AA 5D1.3VH FWR2 402 AA 5D1.3VH CDR2 403 AA5D1.3VH FWR3 404 AA 5D1.4VH FWR1 405 AA 5D1.4VH FWR2 406 AA 5D1.4VH CDR2407 AA 5D1.4VH FWR3 408 AA 5D1.5VH FWR1 409 AA 5D1.5VH FWR2 410 AA5D1.5VH CDR2 411 AA 5D1.5VH FWR3 412 AA 5D1.6VH FWR1 413 AA 5D1.6VH FWR2414 AA 5D1.6VH CDR2 415 AA 5D1.6VH FWR3 416 AA 5D1.7VH FWR1 417 AA5D1.7VH FWR2 418 AA 5D1.7VH CDR2 419 AA 5D1.7VH FWR3 420 AA 5D1.8VH FWR1421 AA 5D1.8VH FWR2 422 AA 5D1.8VH CDR2 423 AA 5D1.8VH FWR3 424 AA5D1.9VH FWR1 425 AA 5D1.9VH FWR2 426 AA 5D1.9VH CDR2 427 AA 5D1.9VH FWR3428 AA 5D1.10VH FWR1 429 AA 5D1.10VH FWR2 430 AA 5D1.10VH CDR2 431 AA5D1.10VH FWR3 432 AA 5D1.11VH FWR1 433 AA 5D1.11VH FWR2 434 AA 5D1.11VHCDR2 435 AA 5D1.11VH FWR3 436 AA 5D1.1VL FWR1 437 AA 5D1.1VL CDR1 438 AA5D1.1VL FWR2 439 AA 5D1.1VL CDR2 440 AA 5D1.1VL FWR3 441 AA 5D1.1VL CDR3442 AA 5D1.1VL FWR4 443 AA 5D1.2VL FWR1 444 AA 5D1.2VL FWR2 445 AA5D1.2VL FWR3 446 AA 5D1.2VL FWR4 447 AA 5D1.3VL FWR1 448 AA 5D1.3VL FWR2449 AA 5D1.3VL FWR3 450 AA 5D1.3VL FWR4 451 AA 5D1.4VL FWR1 452 AA5D1.4VL FWR2 453 AA 5D1.4VL FWR3 454 AA 5D1.4VL FWR4 455 AA 5D1.5VL FWR1456 AA 5D1.5VL FWR2 457 AA 5D1.5VL FWR3 458 AA 5D1.5VL FWR4 459 AA5D1.6VL FWR1 460 AA 5D1.6VL FWR2 461 AA 5D1.6VL FWR3 462 AA 5D1.6VL FWR4463 AA 5D1.7VL FWR1 464 AA 5D1.7VL FWR2 465 AA 5D1.7VL FWR3 466 AA5E8.1VH FWR1 467 AA 5E8.1VH CDR2 468 AA 5E8.1VH FWR3 469 AA 5E8.1VH CDR3470 AA 5E8.2VH FWR1 471 AA 5E8.2VH CDR2 472 AA 5E8.3VH FWR1 473 AA5E8.3VH CDR2 474 AA 5E8.4VH FWR1 475 AA 5E8.4VH CDR2 476 AA 5E8.5VH FWR1477 AA 5E8.5VH CDR2 478 AA 5E8.6VH FWR1 479 AA 5E8.6VH CDR2 480 AA5E8.7VH FWR1 481 AA 5E8.7VH CDR2 482 AA 5E8.8VH FWR1 483 AA 5E8.8VH CDR2484 AA 5E8.9VH FWR1 485 AA 5E8.9VH CDR2 486 AA 5E8.10VH FWR1 487 AA58E.10VH CDR2 488 AA 5E8.11VH FWR1 489 AA 58E.11VH CDR2 490 AA 5E8.1VLFWR1 491 AA 5E8.1VL CDR1 492 AA 5E8.1VL FWR2 493 AA 5E8.1VL CDR2 494 AA5E8.1VL FWR3 495 AA 5E8.1VL CDR3 496 AA 5E8.1VL FWR4 497 AA 5E8.2VL FWR1498 AA 5E8.2VL FWR2 499 AA 5E8.2VL FWR3 500 AA 5E8.2VL FWR4 501 AA5E8.3VL FWR1 502 AA 5E8.3VL FWR2 503 AA 5E8.3VL FWR3 504 AA 5E8.3VL FWR4505 AA 5E8.4VL FWR1 506 AA 5E8.4VL FWR2 507 AA 5E8.4VL FWR3 508 AA5E8.4VL FWR4 509 AA 5E8.5VL FWR1 510 AA 5E8.5VL FWR2 511 AA 5E8.5VL FWR3512 AA 5E8.5VL FWR4 513 AA 10A2.1VH FWR1 514 AA 10A2.1VH CDR1 515 AA10A2.1VH FWR2 516 AA 10A2.1VH CDR2 517 AA 10A2.1VH FWR3 518 AA 10A2.1VHCDR3 519 AA 10A2.1VH FWR4 520 AA 10A2.2VH FWR2 521 AA 10A2.3VH FWR2 522AA 10A2.4VH FWR2 523 AA 10A2.5VH FWR2 524 AA 10A2.6VH FWR2 525 AA10A2.7VH FWR2 526 AA 10A2.8VH CDR1 527 AA 10A2.9VH CDR1 528 AA 10A2.10VHCDR1 529 AA 10A2.11VH CDR1 530 AA 10A2.12VH FWR3 531 AA 10A2.13VH FWR3532 AA 10A2.14VH FWR3 533 AA 10A2.15VH FWR3 534 AA 10A2.16VH CDR3 535 AA10A2.17VH CDR3 536 AA 10A2.18VH CDR3 537 AA 10A2.19VH FWR1 538 AA10A2.19VH FWR2 539 AA 10A2.19VH CDR2 540 AA 10A2.19VH FWR3 541 AA10A2.19VH FWR4 542 AA 10A2.20VH FWR1 543 AA 10A2.20VH FWR2 544 AA10A2.20VH CDR2 545 AA 10A2.20VH FWR3 546 AA 10A2.20VH FWR4 547 AA10A2.21VH FWR1 548 AA 10A2.21VH FWR2 549 AA 10A2.21VH CDR2 550 AA10A2.21VH FWR3 551 AA 10A2.21VH FWR4 552 AA 10A2.22VH FWR1 553 AA10A2.22VH FWR2 554 AA 10A2.22VH CDR2 555 AA 10A2.22VH FWR3 556 AA10A2.22VH FWR4 557 AA 10A2.23VH FWR1 558 AA 10A2.23VH FWR2 559 AA10A2.23VH CDR2 560 AA 10A2.23VH FWR3 561 AA 10A2.23VH FWR4 562 AA10A2.24VH FWR1 563 AA 10A2.24VH FWR2 564 AA 10A2.24VH CDR2 565 AA10A2.24VH FWR3 566 AA 10A2.24VH FWR4 567 AA 10A2.25VH FWR1 568 AA10A2.25VH FWR2 569 AA 10A2.25VH CDR2 570 AA 10A2.25VH FWR3 571 AA10A2.25VH FWR4 572 AA 10A2.26VH FWR1 573 AA 10A2.26VH FWR2 574 AA10A2.26VH CDR2 575 AA 10A2.26VH FWR3 576 AA 10A2.26VH FWR4 577 AA10A2.27VH FWR1 578 AA 10A2.27VH FWR2 579 AA 10A2.27VH CDR2 580 AA10A2.27VH FWR3 581 AA 10A2.27VH FWR4 582 AA 10A2.1VL FWR1 583 AA10A2.1VL CDR1 584 AA 10A2.1VL FWR2 585 AA 10A2.1VL CDR2 586 AA 10A2.1VLFWR3 587 AA 10A2.1VL CDR3 588 AA 10A2.1VL FWR4 589 AA 10A2.2VL CDR1 590AA 10A2.3VL CDR1 591 AA 10A2.4VL CDR2 592 AA 10A2.5VL FWR2 593 AA10A2.6VL FWR2 594 AA 10A2.7VL CDR3 595 AA 10A2.8VL CDR3 596 AA 10A2.9VLCDR3 597 AA 10A2.10VL CDR3 598 AA 10A2.11VL CDR3 599 AA 10A2.12VL CDR3600 AA 10A2.13VL CDR3 601 AA 10A2.14VL CDR3 602 AA 10A2.15VL CDR3 603 AA10A2 16VL CDR3 604 AA 10A2.17VL CDR3 605 AA 10A2.18VL CDR2 606 AA10A2.19VL CDR3 607 AA 10A2.20VL FWR1 608 AA 10A2.20VL CDR1 609 AA10A2,20VL FWR2 610 AA 10A2.20VL CDR2 611 AA 10A2.20VL FWR3 612 AA10A2.20VL CDR3 613 AA 10A2.20VL FWR4 614 AA 10A2.21VL FWR1 615 AA10A2.21VL FWR2 616 AA 10A2.21VL FWR3 617 AA 10A2.21VL FWR4 618 AA10A2.22VL FWR1 619 AA 10A2.22VL FWR2 620 AA 10A2.22VL FWR3 621 AA10A2.22VL FWR4 622 AA 10A2.23VL FWR1 623 AA 10A2.23VL FWR2 624 AA10A2.23VL FWR3 625 AA 10A2.23VL FWR4 626 AA 10A2.24VL FWR1 627 AA10A2.24VL FWR2 628 AA 10A2.24VL FWR3 629 AA 10A2.24VL FWR4 630 AA10A2.25VL FWR1 631 AA 10A2.25VL FWR2 632 AA 10A2.25VL FWR3 633 AA10A2.25VL FWR4 634 AA 10A2.26VL FWR1 635 AA 10A2.26VL FWR2 636 AA10A2.26VL FWR3 637 AA 10A2.26VL FWR4 638 AA 10A2.27VL FWR1 639 AA10A2.27 VL FWR2 640 AA 10A2.27VL FWR3 641 AA 10A2.27VL FWR4 642 AAGly4Ser1 643 AA Gly4Ser1 x 2 644 AA Gly4Ser1 x 3 645 AA Gly4Ser1 x 4 646AA Gly4Ser1 x 5 647 AA IFN-alpha2b A145D 648 AA Trunc IFN-alpha2b 649 AATrunc IFN-alpha2b A145D 650 AA IFN-alpha2b A145G 651 AA TruncIFN-alpha2b A145G 652 AA IgG4 IFN-alpha2b A145D 653 AA IgG4 IFN-alpha2bA145G 654 AA IgG4 S228P IFN-alpha2b A145G 655 AA IgG1 IFN-alpha2b A145G656 AA IgG1 YTE IFN-alpha2b A145D 657 AA IgG1 YTE IFN-alpha2b A145G 658AA IgG4 YTE IFN-alpha2b A145D 659 AA A02 consensus variable heavy 660 AAX02.114VL 661 AA X02.115VL 662 AA X02.116VL 663 AA X02.117VL 664 AA A02consensus variable light 665 AA A10 consensus variable heavy 666 AA A10consensus variable light 667 DNA A02.12VH 668 DNA X02.9VH 669 DNAX02.107VL 670 DNA A02.47VL 671 DNA A02.31VL 672 DNA A02.33VL 673 DNAX02.16VL 674 DNA X02.17VL 675 DNA X02.114VL 676 DNA X02.115VL 677 DNAX02.116VL 678 DNA X02.117VL 679 DNA 10A2VH + A40E 680 DNA 10A2VH + A40G681 DNA 10A2VH + A40H 682 DNA 10A2VH + A40Q 683 DNA 10A2VH + K96G 684DNA 10A2VH + K96T 685 DNA 10A2_1-f*01VH94R 686 DNA 10A2VH (AQ) + N98Q687 DNA 10A2-1-24*0171A94R 688 DNA 10A2_1-33*01Vk 689 DNA 10A2VK + K24G690 DNA 10A2VK + K24Q 691 DNA 10A2VK + R54D 692 DNA 10A2VK + M89A 693DNA 10A2VK (AV) + N53Q 694 AA IgG4 YTE IFN-alpha2b A145G 695 DNA 910VHvariable heavy chain 696 AA 10A2VK + K24G CDR1 697 AA 910VH CDR1 698 AA910VH CDR2 699 AA 910VH CDR3 700 AA X02.118 variable light chain 701 AAX02.119 variable light chain 702 DNA X02.118 variable light chain 703DNA X02.119 variable light chain 704 AA X10.60 variable light chain 705AA X10.61 variable light chain 706 AA X10.62 variable light chain 707 AAX10.63 variable light chain 708 AA X10.64 variable light chain 709 AAX10.65 variable light chain 710 AA X10.66 variable light chain 711 AAX10.67 variable light chain 712 DNA X10.60 variable light chain 713 DNAX10.61 variable light chain 714 DNA X10.62 variable light chain 715 DNAX10.63 variable light chain 716 DNA X10.64 variable light chain 717 DNAX10.65 variable light chain 718 DNA X10.66 variable light chain 719 DNAX10.67 variable light chain 720 AA X10.68 variable heavy chain 721 AAX10.69 variable heavy chain 722 AA X10.70 variable heavy chain 723 AAX10.71 variable heavy chain 724 DNA X10.68 variable heavy chain 725 DNAX10.69 variable heavy chain 726 DNA X10.70 variable heavy chain 727 DNAX10.71 variable heavy chain 728 AA X02.120 variable heavy chain 729 AAX02.121 variable heavy chain 730 AA X02.122 variable heavy chain 731 AAX02.123 variabel heavy chain 732 DNA X02.120 variable heavy chain 733DNA X02.121 variable heavy chain 734 DNA X02.122 variable heavy chain735 DNA X02.123 variable heavy chain 736 AA 910 variable heavy consensus737 AA X02.122VH CDR2 738 AA X02.123VH CDR2 739 AA X10.72 variable heavychain 740 AA X10.73 variable heavy chain 741 AA X10.74 variable heavychain 742 AA X10.75 variable heavy chain 743 DNA X10.72 variable heavychain 744 DNA X10.73 variable heavy chain 745 DNA X10.74 variable heavychain 746 DNA X10.75 variable heavy chain 747 AA X10.64VL CDR2 748 AA910 VH FRW1 749 AA 910 VH FRW2 750 AA X10.120VH FRW2 751 AA 910 VH FRW3752 AA X10.121VH FRW3 753 AA 910VH FRW4

The disclosure is not limited to the embodiments described andexemplified above, but is capable of variation and modification withinthe scope of the appended claims.

We claim:
 1. A polynucleotide comprising a nucleic acid sequenceencoding a recombinant antibody, the recombinant antibody comprising theheavy chain complementarity determining region 1 (CDR1), CDR2, and CDR3of SEQ ID NO: 665 and the light chain CDR1, CDR2, and CDR3 of SEQ ID NO:666, wherein the antibody specifically binds to CD38.
 2. Thepolynucleotide of claim 1, wherein the recombinant antibody is ahumanized antibody.
 3. The polynucleotide of claim 1, wherein theantibody comprises a human IgG1 heavy chain constant region.
 4. Thepolynucleotide of claim 1, wherein the antibody comprises a human IgG4heavy chain constant region.
 5. The polynucleotide of claim 4, whereinthe human IgG4 heavy chain constant region comprises a proline atposition 228 according to the EU numbering system.
 6. The polynucleotideof claim 1, wherein the nucleic acid sequence encodes an attenuatedhuman interferon alpha-2b fused to the antibody.
 7. The polynucleotideof claim 6, wherein the attenuated human interferon alpha-2b comprisesan alanine to aspartic acid substitution at position 145 relative to theamino acid sequence of SEQ ID NO:
 648. 8. The polynucleotide of claim 6,wherein the attenuated human interferon alpha-2b comprises the aminoacid sequence of SEQ ID NO: 647, SEQ ID NO: 649, SEQ ID NO: 650, or SEQID NO:
 651. 9. The polynucleotide of claim 1, comprising a nucleic acidsequence comprising SEQ ID NO: 685, which encodes a heavy chain variableregion, and a nucleic acid sequence comprising SEQ ID NO: 691, whichencodes a light chain variable region.
 10. A vector comprising thepolynucleotide of claim
 1. 11. A transformed mammalian cell comprisingthe vector of claim
 10. 12. A nucleic acid encoding a recombinantantibody, the antibody comprising a heavy chain variable regioncomprising a CDR1, CDR2, and CDR3, the heavy chain variable regioncomprising an amino acid sequence that is at least 95% identical to theamino acid sequence of SEQ ID NO: 156 and a light chain variable regioncomprising a CDR1, CDR2, and CDR3, the light chain variable regioncomprising an amino acid sequence that is at least 95% identical to theamino acid sequence of SEQ ID NO: 185, wherein any amino acid changes inthe heavy chain variable region or light chain variable region areoutside of the CDRs
 13. The nucleic acid of claim 12, wherein theantibody comprises a heavy chain variable region comprising the aminoacid sequence of SEQ ID NO: 156 and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO:
 185. 14. The nucleicacid of claim 12, wherein the nucleic acid further encodes an attenuatedhuman interferon alpha-2b fused to the antibody.
 15. The nucleic acid ofclaim 14, wherein the attenuated human interferon alpha-2b comprises analanine to aspartic acid substitution at position 145 relative to theamino acid sequence of SEQ ID NO: 648
 16. A method for treating a tumorin a subject, the method comprising: administering to the subject arecombinant antibody that specifically binds to CD38, the recombinantantibody comprising the heavy chain complementarity determining region 1(CDR1), CDR2, and CDR3 of SEQ ID NO: 665 and the light chain CDR1, CDR2,and CDR3 of SEQ ID NO: 666 to thereby treat the tumor.
 17. The methodaccording to claim 16, wherein the tumor is a B-cell lymphoma, multiplemyeloma, non-Hodgkin's lymphoma, chronic myelogenous leukemia, chroniclymphocytic leukemia, or acute myelogenous leukemia.
 18. The method ofclaim 16, wherein the recombinant antibody is a humanized antibody. 19.The method of claim 16, wherein the antibody is fused to an attenuatedhuman interferon alpha-2b.
 20. The method of claim 19, wherein theattenuated human interferon alpha-2b comprises an alanine to asparticacid substitution at position 145 relative to the amino acid sequence ofSEQ ID NO: 648.